JP2006518595A - Methods using adenoviral vectors with increased in vivo persistence - Google Patents

Abstract

The present invention provides a method for expressing a foreign nucleic acid in a mammal.
The method includes gradually releasing doses of replication-deficient or conditionally replicating adenoviral vectors with reduced ability to transduce mesothelial cells and hepatocytes into the bloodstream. The normalized mean blood flow concentration of adenovirus over 24 hours after administration is at least about 1%. Also, the normalized mean blood flow concentration over 24 hours after administration is at least about 5 times greater than the normalized mean blood flow concentration of the equivalent dose of wild type adenoviral vector. A method of destroying tumor cells in a mammal is also provided.

Description

(Field of Invention)
The present invention relates to a method of achieving increased adenoviral vector persistence in circulation.

(Background of the Invention)
Gene therapy is gaining acceptance in the scientific community as being useful for the treatment of various diseases. Adenovirus-derived gene transfer vectors have many attractive properties in gene therapy, including substantial and transient gene expression, the ability to grow at high titers, and the ability to transduce a wide variety of cell types. Proven to have Despite these advantageous properties, adenoviral vectors are subject to the same limitations as other gene transfer vectors in achieving a broad range of delivery in the body.

  Viral vectors encode and / or display antigenic epitopes that are naturally recognized by the host immune system. The immunogenicity of viral vectors, including adenoviral vectors, is a major obstacle in the use of these transgenic vehicles in vivo. For example, the majority of the human population is exposed to adenovirus and therefore has pre-existing immunity against adenovirus vectors based on human adenovirus serotypes, which is effective for the virus as a gene transfer vector. Restricts sex. In addition to existing immunity, adenoviral vector administration induces inflammation and activates both innate and acquired immune mechanisms. Adenoviral vectors activate an antigen-specific (eg, T cell-dependent) immune response, which limits the duration of transgene expression after the first administration of the vector. Moreover, exposure to adenoviral vectors stimulates the production of neutralizing antibodies by B cells, which prevents gene expression from subsequent administration of adenoviral vectors (Wilson & Kay, Nat. Med., 3 (9 ), 887-889 (1995)). Indeed, the effectiveness of repeated administration of the vector can be severely limited by host immunity. For example, animal experiments can result in the production of neutralizing antibodies against a vector, where intravenous or local administration of an adenovirus serotype 2 or 5 vector prevents expression from the same serotype vector administered after 1-2 weeks. (See, for example, Kass-Eisler et al., Gene Therapy, 1, 395-402 (1994) and Kass-Eisler et al., Gene Therapy, 3, 154-162 (1996)).

  In addition to stimulating humoral immunity, cell-mediated immune functions are responsible for clearance of the virus from the body. Rapid clearance of the virus is due to innate immune mechanisms (see, eg, Worgall et al., Human Gene Therapy, 8, 37-44 (1997)) and appears to involve Kupffer cells found in the liver . Adenoviral vectors are typically removed from circulation within minutes and removed from the body within about 7-10 days. Within the first two days of infection, approximately 90% of the adenoviral vector DNA is removed (Elkon et al., PNAS, 94, 9814-9819 (1997)). Rapid clearance of adenoviral vectors reduces circulation time and prevents efficient delivery to target cells via systemic circulation that may be necessary to treat diseases such as disseminated cancer.

  To address the shortcomings of adenoviral vectors with regard to persistence in the body, modification of adenoviral particle antigenic determinants has been envisaged. It is inferred that avoiding the body's clearance mechanism will increase the amount of time in the circulation, thereby increasing the likelihood of transducing target cells distal from the point of administration. Adenovirus fiber, penton, and hexon proteins have received the most attention because they represent the initial exposure of the virus to the host's immune and clearance systems. For example, US Pat. No. 6,153,435 (Crystal et al.) Describes an adeno having a chimeric adenovirus coat protein with reduced or unrecognizable ability to be recognized by neutralizing antibodies to the corresponding wild type adenovirus coat protein. Viral vectors have been described. Genetic manipulation of adenovirus coat proteins, although somewhat limited, has resulted in successful evasion of host immunity.

  Despite advances in regulating the antigenicity of adenoviral vectors, improvements in the use of adenoviral vectors in vivo increase the retention of adenoviral vectors in the body, obtain better distribution, and improve trait to target cells. There is a need to increase adoption. The present invention provides methods of using such adenoviral vectors to obtain a sustained increase in circulation. These and other advantages and additional inventive features of the present invention will become apparent from the description of the invention provided herein.

(Simple Summary of Invention)
The present invention provides a method for expressing a foreign nucleic acid in a mammal. The method involves gradually releasing a single dose of a replication-deficient or conditionally replicating adenoviral vector into the mammalian bloodstream. The adenoviral vector has a reduced ability to transduce mesothelial cells and hepatocytes compared to wild type adenovirus. A replication-deficient or conditionally replicating adenoviral vector further comprises a foreign nucleic acid. The normalized mean blood flow concentration of replication deficient or conditionally replicating adenovirus over 24 hours after administration is at least 1%. Alternatively, the normalized mean blood flow concentration of replication deficient or conditionally replicating adenovirus over 24 hours after administration is at least about 5 times greater than the normalized mean blood flow concentration for equivalent doses of wild type adenovirus. Mammalian host cells are transduced with replication deficient or conditionally replicating adenoviral vectors to express foreign nucleic acids.

  The present invention further provides a method of destroying tumor cells in a mammal. The method comprises a single dose of replication deficiency comprising (a) a nucleic acid sequence encoding an oncocidal factor and (b) an adenovirus fiber protein that does not mediate adenovirus entry via coxsackievirus and adenovirus receptor (CAR) Or it involves gradual delivery of a conditionally replicating adenoviral vector into the bloodstream, thereby producing tumorigenic factors and destroying tumor cells in the mammal.

(Detailed description of the invention)
The present invention provides that, at least in part, gene transfer vectors, particularly adenoviral gene transfer vectors, have a larger fraction of a single dose of gene transfer vector for at least 24 hours after administration than previously achieved. It is based on the surprising discovery that it can be delivered to the mammalian systemic circulation to survive. Adenoviral vectors are typically removed from circulation within minutes. Failure to hold an adenoviral vector in circulation limits the effectiveness of a single dose of adenoviral gene transfer vector in delivering the transgene to target cells, particularly target cells distal from the point of administration. For example, the most effective method of delivering a single dose of adenoviral vector to a target tissue is direct injection of the virus into the tissue, whereby the majority of the dose contacts the target cells. However, direct injection into the target tissue cannot be performed if the target tissue cannot be easily reached by injection or if the target cells are scattered throughout the body. The present invention provides a method for delivering an adenoviral gene transfer vector to the mammalian circulatory system for distribution throughout the body, thereby maximizing the dose of the adenoviral vector to increase the likelihood of target cell transduction. As long as possible. Adenoviral vectors that remain in circulation for several minutes, preferably several hours or more after administration, i.e., 1, 3, 5, or 7 days and remain capable of transducing or proliferating cells are in vivo. Is said to have an increased half-life, increased persistence, or increased circulation time.

  In particular, the present invention provides a method for expressing a foreign nucleic acid in a mammal. The method involves gradually releasing a single dose of a replication-deficient or conditionally replicating adenoviral vector containing the foreign nucleic acid into the mammalian bloodstream. Replication deficient or conditionally replicating adenoviral vectors have a reduced ability to transduce mesothelial cells and hepatocytes compared to wild type adenovirus. The normalized mean blood flow concentration of replication deficient or conditionally replicating adenovirus over 24 hours after administration is at least 1%. Mammalian host cells are transduced and foreign nucleic acid is expressed in the cells.

Adenovirus vectors Any source of adenovirus, any subtype, mixture of subtypes, or any chimeric adenovirus can be used as the viral genome source of a replication defective or conditionally replicating adenoviral vector. Although non-human adenoviruses (eg, monkeys, birds, dogs, sheep, or bovine adenoviruses) can be used to generate replication-deficient adenovirus vectors, preferably human adenoviruses are replicated in the methods of the invention. Used as a viral genome source for defective or conditionally replicating adenoviral vectors. The adenoviral vector may be of any subgroup or serotype. For example, adenoviruses are subgroup A (eg, serotypes 12, 18, and 31), subgroup B (eg, serotypes 3, 7, 11, 14, 16, 21, 34, 35, and 50), subgroups C (eg, serotypes 1, 2, 5, and 6), subgroup D (eg, serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36- 39, and 42-48), subgroup E (eg, serotype 4), subgroup F (eg, serotypes 40 and 41), unclassified serogroup (eg, serotypes 49 and 51), or It can be of any other adenovirus serotype. Adenovirus serotypes 1 to 51 are available from the American Type Culture Collection (ATCC, Manassas, VA). Preferably, the adenoviral vector is human subgroup C, particularly serotype 2 or more desirably serotype 5. Serotype 35 or serotype 41 adenoviral vectors are also suitable for use in the present invention.

  “Replication deficient” means that the adenoviral vector contains an adenoviral genome that lacks at least one gene function essential for replication (ie, the adenoviral vector is in accordance with the present invention, in particular a normal host cell). Do not replicate in cells in human patients who would be able to infect adenoviral vectors during the course of therapy). A gene, gene function, or deletion in a gene region or genomic region as used herein is a lack of sufficient genetic material in the viral genome that diminishes or destroys the function of the gene in which all or part of its nucleic acid sequence has been deleted. Defined as lost. Gene functions essential for replication are those required for replication (eg, proliferation), such as adenovirus early region (eg, E1, E2, and E4 regions), late region (eg, L1-L5 region). , Encoded by genes involved in viral packaging (eg, the IVa2 gene), and virus-related RNAs (eg, VA-RNA1 and / or VA-RNA-2). More preferably, the replication defective adenoviral vector comprises an adenoviral genome that is deficient in at least one gene function essential for replication in one or more regions of the adenoviral genome. Preferably, the adenoviral vector is deficient in at least one gene function of the E1 region of the adenoviral genome necessary for viral replication (denoted as E1-deficient adenoviral vector). In addition to defects in the E1 region, recombinant adenoviruses can also have mutations in the major late promoter (MLP), as discussed in International Patent Application WO 00/00628. Most preferably, the adenoviral vector has a gene function essential for at least one replication of the E1 region (preferably a gene function essential for all replications) and at least a portion of the non-essential E3 region (eg, Xba I deficiency of the E3 region). (Denoted E1 / E3-deficient adenovirus vector). With respect to the E1 region, the adenoviral vector may lack a gene function that is essential for at least one replication of part or all of the E1A region and part or all of the E1B region, eg, each of the E1A and E1B regions. When an adenoviral vector lacks at least one gene function essential for replication in one region of the adenoviral genome (eg, an E1- or E1 / E3-deficient adenoviral vector), the adenoviral vector is designated as “single replication”. It is called “deficiency”. Particularly preferred single replication deficient adenoviral vectors are those described in the Examples herein.

  The adenoviral vector may be “multiple replication deficient” meaning that one or more of the two or more regions of the adenoviral genome are deficient in one or more gene functions essential for replication. For example, the above-mentioned E1-deficient or E1 / E3-deficient adenoviral vector is the E4 region (denoted as E1 / E4- or E1 / E3 / E4-deficient adenoviral vector) and / or the E2 region (E1 / E2- Or an E1 / E2 / E3-deficient adenoviral vector), preferably at least one gene function essential for replication of the E2A region (denoted E1 / E2A- or E1 / E2A / E3-deficient adenoviral vector) Can be deficient. Ideally, adenoviral vectors lack the gene function essential for replication of only the gene function essential for replication encoded in the early region of the adenoviral genome, however, this is the case for all situations of the present invention. Is not necessary. Preferred multi-deficient adenoviral vectors are nucleotides 457-3332 of the E1 region, nucleotides 28593-30470 of the E3 region, nucleotides 32826-35561 of the E4 region, and optionally nucleotides 10594-10595 of the region encoding VA-RNA1. Includes adenoviral genomes with deletions. However, other deletions may be appropriate. Nucleotides 356-3329 or 356-3510 can be removed to create a defect in the E1 gene function essential for replication. Nucleotides 28594-30469 can be deleted from the E3 region of the adenovirus genome. Although the specific nucleotide designations listed above correspond to the adenovirus serotype 5 genome, the corresponding nucleotides for the non-serotype 5 adenovirus genome can be readily determined by one skilled in the art.

  Adenoviral vectors are complementary, particularly in the case of multiple replication deficiencies in gene functions essential for replication of the E1 and E4 regions, preferably as in single replication deficient adenoviral vectors, particularly E1-deficient adenoviral vectors. Contains a spacer element to provide viral propagation in the cell line. The spacer element can comprise any sequence or sequences of the desired length, eg, the sequence is at least about 15 base pairs (eg, between about 15 base pairs and about 12,000 base pairs), preferably about 100 base pairs to about 10,000 base pairs, more preferably about 500 base pairs to about 8,000 base pairs, more preferably about 1,500 base pairs to about 6,000 base pairs, most preferably about 2,000. ~ About 3,000 base pairs in length. The spacer element sequence can be coding or non-coding and native or non-native with respect to the adenoviral genome, but does not restore the function essential for replication of the defective region. In the absence of spacers, the production of fiber protein and / or viral propagation of a multiple replication defective adenoviral vector is reduced compared to that of a single replication defective adenoviral vector. However, the introduction of a spacer into at least one of the defective adenoviral regions (preferably the E4 region) can prevent this reduction in fiber protein production and viral growth. The use of spacers in adenoviral vectors is described in US Pat. No. 5,851,806. In one embodiment of the method of the invention, the replication-deficient or conditionally-replicating adenoviral vector has an E1 / E4-deficiency in which the L5 fiber region is retained and a spacer is located between the L5 fiber region and the right ITR. Adenovirus vector. More preferably, in such adenoviral vectors, the E4 polyadenylation sequence is present alone or, most preferably, in combination with other sequences, is present and retained between the L5 fiber region and the right ITR. The L5 fiber region is well separated from the right ITR, so that the viral production of such vectors approaches that of single replication defective adenoviral vectors, particularly E1-deficient adenoviral vectors.

  The adenoviral vector may lack gene functions essential for replication of only the early region of the adenoviral genome, only the late region of the adenoviral genome, and both the early and late regions of the adenoviral genome. Adenoviral vectors may also have substantially the entire adenoviral genome removed, in which case at least viral inverted terminal repeats (ITRs) and one or more promoters, or viral ITRs and packaging signals (ie, Preferably, either one of the adenovirus amplicons) is intact. The adenoviral genome 5 'or 3' region containing the ITRs and packaging sequences need not be from the same adenoviral serotype as the rest of the viral genome. For example, the 5 ′ region of the adenovirus serotype 5 genome (ie, the 5 ′ to the adenovirus E1 region genomic region) is replaced with the corresponding region of the adenovirus serotype 2 genome (eg, 5 ′ to the adenovirus genome The Ad5 genomic region of the E1 region may be replaced with nucleotides 1-456 of the Ad2 genome). Suitable replication-deficient adenoviral vectors, including multiple replication-deficient adenoviral vectors are US Pat. Nos. 5,837,511; 5,851,806; 5,994,106; and 6,127,175; US Published Patent Applications 2001/0043922 A1; 2002/0004040 A1; / 0110545 A1, and international patent applications WO 94/28152; WO 95/02697; WO 95/34671; WO 96/22378; WO 97/12986; and WO 97/21826. Ideally, replication-deficient or conditionally replicating adenoviral vectors are adenoviral vector compositions, particularly pharmaceutical compositions that are substantially free of replication-competent adenovirus (RCA) (eg, the composition is about 1 Used in the present invention in the form of less than% RCA contamination). Most desirably, the composition is free of RCA contamination. Adenoviral vector compositions and stock free of RCA are described in US Pat. Nos. 5,944,106 and 6,482,616, US Published Patent Application 2002/0110545 Al, and International Patent Application WO 95/34671.

  A replication-deficient adenoviral vector is usually not present in a replication-deficient adenoviral vector to produce a highly infectious viral vector stock, but a complementary cell line that provides the gene function necessary for viral growth at an appropriate level Produced in. Preferred cell lines complement at least one, preferably all replication essential, gene function that is not present in replication-defective adenoviruses. Complementary cell lines are encoded in the early region, late region, viral packaging region, virus-associated RNA region, or combinations thereof, and are responsible for all adenoviral functions (eg, allowing for growth of adenoviral amplicons). It can complement a deficiency in gene function essential for at least one replication. Most preferably, the complementary cell line is deficient in a gene function essential for at least one replication of the E1 region of the adenovirus genome (eg, a gene function essential for two or more replications), particularly for each replication of the E1A and E1B regions. Complements essential gene function deficiency. In addition, the complementary cell line may complement a deficiency in gene function essential for at least one replication of the E2 (especially for adenovirus DNA polymerase and terminal proteins) and / or E4 regions of the adenovirus genome. Desirably, the cell that complements the E4 region defect comprises the E4-ORF6 gene sequence and produces an E4-ORF6 protein. Such cells desirably contain at least ORF6 and no other ORFs of the E4 region of the adenovirus genome. The cell line is preferably characterized by containing a complementary gene so that it does not overlap with the adenoviral vector, which minimizes and virtually eliminates the possibility that the vector genome will recombine with the DNA of the cell. Thus, the presence of replicative adenovirus (RCA) is unavoidably minimized in vector stocks and is therefore suitable for certain therapeutic purposes, particularly gene therapy purposes. RCA deficiency in vector stocks avoids adenoviral vector replication in non-complementing cells. Construction of such complementary cell lines includes standard molecular biology and cell culture techniques such as Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d edition, Cold Spring Harbor Press, Cold Spring Harbor, NY ( 1989), and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, NY (1994).

  Complementary cell lines for producing adenoviral vectors include, but are not limited to, 293 cells (eg, as described in Graham et al., J. Gen. Virol., 36, 59-72 (1977)), PER.C6 cells (For example, described in International Patent Application WO 97/00326 and US Patents 5,994,128 and 6,033,908), and 293-ORF6 cells (for example, International Patent Application WO 95/34671 and Brough et al., J. Virol., 71, 9206). -9213 (1997)). In some cases, the complementing cells will not complement all necessary adenoviral gene functions. Helper viruses can be used to provide in gene functions that are not encoded in the cell or adenoviral genome to allow replication of adenoviral vectors. Adenoviral vectors are described, for example, in U.S. Pat.Nos. Constructed and propagated using materials and methods shown in / 56937, WO 99/15686, WO 99/54441, WO 00/12765, WO 01/77304, and WO 02/29388, and other references identified herein And / or purified. Non-C group adenovirus vectors, including adenovirus serotype 35 vectors, can be produced using, for example, the methods set forth in US Pat. Nos. 5,837,511 and 5,849,561, and international patent applications WO 97/12986 and WO 98/53087. In addition, many adenoviral vectors are commercially available.

  If the adenoviral vector is not replication defective, ideally the adenoviral vector is engineered to limit replication of the vector in the target tissue. For example, the adenoviral vector can be a conditionally replicating adenoviral vector that has been engineered to replicate under conditions predetermined by the practitioner. For example, a gene function essential for replication, such as the gene function encoded by the adenovirus early region, can be operably linked to an inducible, repressible, or tissue-specific transcriptional regulatory sequence, such as a promoter. In this embodiment, replication requires the presence or absence of specific factors that interact with transcriptional regulatory sequences. Conditionally replicating adenoviral vectors are particularly useful for delivering foreign nucleic acids for the purpose of destroying target cells. Adenoviral vector replication can be limited to the target tissue, which allows greater distribution of the vector throughout the tissue while effectively utilizing the natural ability of adenovirus to lyse cells during the replication cycle. Become. In cancer therapy, conditionally replicating adenoviruses provide a method of destroying tumor cells in addition to delivering lethal foreign nucleic acids. Conditionally replicating adenoviral vectors are further described in US Pat. No. 5,998,205.

  Replication deficient or conditionally replicating adenoviral vectors have a reduced ability to transduce mesothelial cells and hepatocytes compared to wild-type adenoviruses of the same serotype as replication deficient or conditionally replicating adenoviral vectors . Adenoviruses that do not naturally transduce mesothelial cells and hepatocytes, such as some non-human adenoviruses, can be used in the present invention. However, adenoviral vectors based on serotypes of human adenovirus that naturally infect mesothelial and liver cells are modified to reduce binding to these cells. “Decreased” transduction or binding is a serum in which the level of transduction by a replication-deficient or conditionally replicating adenoviral vector of a target cell such as a mesothelial cell or hepatocyte is the same as that of a replication-deficient or conditionally replicating adenoviral vector. At least approximately 3 times less than the level of transduction mediated by the wild type adenovirus (eg, at least approximately 5 times, 10 times, 15 times, 20 times, 25 times, 35 times, 45 times, or 50 times) Less). Preferably, the decrease in transduction efficiency is a substantial decrease (such as at least an order of magnitude, preferably more). Desirably, replication-deficient or conditionally replicating adenoviral vectors do not transduce mesothelial cells or hepatocytes.

  To reduce the native binding and transduction of replication-deficient or conditionally-replicating adenoviral vectors, the native binding sites located on adenoviral coat proteins that mediate cell entry, such as fiber and / or penton bases, are missing. Or destroyed. Two or more adenovirus coat proteins are believed to mediate cell surface adhesion (eg, fiber and penton based). Any suitable technique that alters native binding to host cells (eg, mesothelial cells or hepatocytes) can be used. For example, utilizing different fiber lengths to eliminate binding to native cells can be accomplished through the addition of binding sequences to the penton base or fiber knob. This addition can be made either directly or indirectly through a bispecific or multispecific binding sequence. Alternatively, the adenovirus fiber protein can be modified to reduce the number of amino acids in the fiber shaft, thereby creating a “short shaft” fiber (eg, as described in US Pat. No. 5,962,311). Some adenovirus serotype fiber proteins are inherently shorter than others and can be used to replace these fiber proteins with native fiber proteins to allow the native binding of adenovirus to its native receptor. Can be reduced. For example, the native fiber protein of an adenovirus vector derived from serotype 5 adenovirus can be replaced with a fiber protein derived from adenovirus serotype 40 or 41.

  In other embodiments, by mutating nucleic acid residues associated with native substrate binding (eg, International Patent Application WO 00/15823; Einfeld et al., J. Virol., 75 (23), 11284-11291 (2001); and van Beusechem et al., J. Viol., 76 (6), 2753-2762 (2002)) The ability of adenoviral vectors incorporating mutated nucleic acid residues to bind to their native substrates Can be reduced. For example, adenovirus serotypes 2 and 5 transduce cells via binding of adenovirus fiber protein to Coxsackievirus and adenovirus receptor (CAR) and binding of penton protein to integrins located on the cell surface. . Thus, the replication-deficient or conditionally replicating adenoviral vectors of the methods of the invention can lack native binding to CAR and / or exhibit reduced native binding to integrin. In order to reduce the native binding of the replication-deficient or conditionally-replicating adenoviral vector to the host cell, the native CAR and / or integrin binding sites (eg, the RGD sequence located in the adenoviral penton base) are removed or destroyed. The

  A replication-deficient or conditionally-replicating adenoviral vector can also include a chimeric coat protein comprising a non-native amino acid sequence that binds to a substrate (ie, a ligand). Since the method of the present invention allows adenoviral vectors to remain in circulation for an extended period of time, the method of the present invention preferentially includes a “targeted” adeno that contains a non-native amino acid sequence that binds to a target cell preferentially. Particularly suitable for the use of viral vectors. The non-native amino acid sequence of the chimeric adenovirus coat protein is such that the adenoviral vector containing the chimeric coat protein is not naturally infected by the corresponding adenovirus without the non-native amino acid sequence (ie, the corresponding wild type Host cells that are not infected by a type of adenovirus), desirably infecting it naturally, with a greater affinity than the corresponding adenovirus without a native amino acid sequence, and naturally infecting with the corresponding adenovirus Allowing binding to a specific target cell, or with greater affinity than non-target cells. A “non-native” amino acid sequence can include an amino acid sequence that does not naturally occur in an adenovirus coat protein or an amino acid sequence that is found in the adenovirus coat but located at a non-native position within the capsid. “Preferentially binds” means that the non-native amino acid sequence has an affinity (eg, at least about 5-fold greater) than the non-native ligand binds to a different receptor, eg, αvβ1 integrin. Means binding with a receptor such as αvβ3 integrin, for example, with a 10-fold, 15-fold, 20-fold, 25-fold, 35-fold, 45-fold or 50-fold greater affinity).

  The non-native amino acid sequence can be conjugated to any adenovirus coat protein to form a chimeric coat protein. Thus, for example, a non-native amino acid sequence of the invention can be conjugated, inserted or attached to a fiber protein, penton base protein, hexon protein, protein IX, VI, or IIIa, etc. Sequences of such proteins and methods for using them in recombinant proteins are well known in the art (eg, US Pat. Nos. 5,543,328; 5,559,099; 5,712,136; 5,731,190; 5,756,086; 5,770,442; 5,846,782; 5,962,311; 5,965,541 5,846,782; 6,057,155; 6,127,525; 6,153,435; 6,329,190; 6,455,314; 6,465,253; and 6,576,456; US Patent Application Publications 2001/0047081 and 2003/0099619; and International Patent Applications WO 96/07734, WO 96/26281, WO 97/20051, WO 98/07877, WO 98/07865, WO 98/40509, WO 98/54346, WO 00/15823, WO 01/58940, and WO 01/92549). The coat protein portion of the chimeric coat protein may be a full length adenovirus coat protein to which a ligand domain is added, or may be truncated, for example, on the inside or at the C and / or N terminus. The outer protein portion itself need not be native to the adenoviral vector. For example, the coat protein can be an adenovirus serotype 4 (Ad4) fiber protein incorporated into an adenovirus serotype 5 vector, where the native CAR binding motif of the Ad4 fiber is preferably removed. Similarly, simian adenovirus type 25 (SAV-25) fiber protein can be incorporated into the adenovirus serotype 35 capsid. The native binding of SAV-25 fiber can be removed by mutating the AB loop and β-sheet of the fiber protein, and optionally, the non-native amino acid sequence is inserted into the H1 loop of the fiber protein or attached to the C-terminus. obtain. No matter how modified (including the presence of non-native amino acids), the chimeric coat protein can preferably be incorporated into the adenovirus capsid as its native corresponding coat protein. Once a given non-native amino acid sequence is identified, it can be incorporated at any location on the virus that can interact with the substrate (ie, the viral surface). For example, the ligand can be incorporated into a fiber, penton base, hexon, protein IX, VI, or IIIa, or other suitable location. When the ligand is attached to the fiber protein, it preferably does not interfere with the interaction between viral proteins or fiber monomers. Thus, it is preferred that the non-native amino acid sequence is not itself an oligomerization domain because it can interact adversely with the trimerization domain of adenovirus fiber. Preferably, the ligand is added to the virion protein so that it is easily exposed to the substrate to maximize the display of the non-native amino acid sequence to the substrate (eg, at the N or C terminus of the protein, the substrate Attached to the residue facing the substrate and positioned on the peptide spacer in contact with the substrate, etc.). Ideally, the non-native amino acid sequence is incorporated into the adenoviral fiber protein at the C-terminus of the fiber protein (and attached via a spacer) or into an exposed loop of the fiber (eg, a HI loop) Produces a chimeric shell protein. If the non-native amino acid sequence is attached to or replaced by a part of the penton base, preferably it is in the hypervariable region to ensure contact with the substrate. If the non-native amino acid sequence is attached to a hexon, preferably it is in the hypervariable region (Miksza et al., J. Virol., 70 (3), 1836-44 (1996)). The use of a spacer sequence to extend the non-native amino acid sequence away from the surface of the adenovirus particle makes it possible for the non-native amino acid sequence to be available upon binding to the receptor, between the non-native amino acid sequence and the adenovirus fiber monomer. It can be advantageous in that any steric interaction is reduced.

  The binding affinity of a non-native amino acid sequence to a cellular receptor can be measured by any suitable assay, and a variety of such assays are known and include non-native amino acid sequences for incorporation into adenovirus coat proteins. Useful for choosing. Desirably, the level of transduction of the host cell is utilized to measure relative binding efficiency. Thus, for example, host cells that display αvβ3 integrin on the cell surface (eg, MDAMB435 cells) can be transformed into a replication-deficient or conditionally replicating adenoviral vector containing a chimeric coat protein and a corresponding adenovirus without a non-native amino acid sequence. After exposure to, the transduction efficiency can be compared to determine the relative binding affinity. Similarly, a host cell that presents αvβ3 integrin on the cell surface (eg, MDAMB435 cell) and a host cell that primarily presents αvβ1 on the cell surface (eg, 293 cell) are combined with adenos containing chimeric outer shell proteins. After exposure to the viral vector, the transduction efficiency can be compared to determine binding affinity.

  A non-native amino acid sequence binds to a specific cell receptor present in a narrow group of cell types (eg, tumor cells, myocardium, skeletal muscle, smooth muscle, etc.) or a wider group that includes several cell types Can do. By incorporating appropriate cell-specific ligands, viral particles can be used, for example, neuronal cells, glial cells, endothelial cells (eg, tissue factor receptor, FLT-1, CD31, CD36, CD34, CD105, CD13, ICAM -1 (McCormick et al., J. Biol. Chem., 273, 26323-29 (1998)), thrombomodulin receptor (Lupus et al., Suppl., 2, S 120 (1998)), VEGFR-3 ( Lymboussaki et al., Am. J. Pathol., 153 (2), 395-403 (1998), mannose receptor, VCAM-1 (Schwarzacher et al., Atherocsclerosis, 122, 59-67 (1996)), or (Via other receptors), blood clots (eg by fibrinogen or aIIbb3 peptide), epithelial cells (eg inflammatory tissue via sorting of VCAM-1, ICAM-1, etc.), keratinocytes, follicular cells, adipocytes, Fibroblasts, hematopoietic or other stem cells, myoblasts, myofibrils, cardiomyocytes, smooth muscle, somatic cells, osteoclasts, osteoblasts, odontocytes, soft Bone cells, melanocytes, hematopoietic cells, etc., and cancer cells derived from any of the above cell types (eg, prostate (such as via the PSMA receptor (eg, 271 (12), 7043-7051 (1996); Cancer Res., 58, 4055 (1998))), breast, lung, brain (eg glioblastoma), leukemia / lymphoma, liver, sarcoma, bone, colon Any desired cell type can be targeted, such as testis, ovary, bladder, throat, stomach, pancreas, rectum, skin (eg, melanoma), kidney, etc.).

  In other embodiments (eg, to facilitate purification or growth in a particular engineered cell type), non-native amino acids (eg, ligands) can bind to compounds other than cell surface proteins. Thus, ligands can be blood and / or lymph-derived proteins (eg, albumin), synthetic peptide sequences such as polyamino acids (eg, polylysine, polyhistidine, etc.), artificial peptide sequences (eg, FLAG), and RGD. It can bind to peptide fragments (Pasqualini et al., J. Cell. Biol., 130, 1189 (1995)). Ligand can be plastic (eg Adey et al., Gene, 156, 27 (1995)), biotin (Saggio et al., Biochem. J., 293, 613 (1993)), DNA sequence (Cheng et al., Gene, 171, 1 (1996); Krook et al., Biochem. Biophys., Res. Commun., 204, 849 (1994)), streptavidin (Geibel et al., Biochemistry, 34, 15430 (1995); Katz , Biochemistry, 34, 15421 (1995)), nitrostreptavidin (Balass et al., Anal. Biochem., 243, 264 (1996)), heparin (Wickham et al., Nature Biotechnol., 14, 1570) -73 (1996)), or even non-peptide substrates such as other possible substrates.

  Examples of non-native amino acid sequences and their substrates suitable for use in the methods of the present invention include, but are not limited to, short (eg, 6 amino acids or less) linearly extended amino acids recognized by integrins, and Examples include polyamino acid sequences such as polylysine and polyarginine. Multiple lysine and / or arginine insertions provide heparin and DNA recognition. Suitable non-native amino acid sequences for generating chimeric adenovirus coat proteins are further described in US Patent 6,455,314 and International Patent Application WO 01/92549.

  Preferably, the adenovirus coat protein comprises a non-native amino acid sequence that binds to αvβ3, αvβ5, or αvβ6 integrin. In order to increase targeting efficiency, the native binding of adenoviral coat proteins to native adenoviral cell surface receptors, such as Coxsackie and adenoviral receptors (CAR), is as described herein. To be removed. Preferably, when a non-native amino acid sequence binds to αvβ3 integrin, the non-native amino acid sequence binds with an affinity that is at least about 10 times greater than that binds to αvβ1 integrin. αvβ3 integrin is upregulated in tumor tissue vasculature, metastatic breast cancer, melanoma, and glioma. Adenoviral vectors that present a ligand specific for αvβ3 integrin, such as the RGD motif, infect cells with more αvβ3 integrin moieties on the cell surface than cells that do not express integrin to that extent. Thereby targeting the vector to the specific cells of interest. In one embodiment, the RGD motif is sandwiched between one or two sets of cysteine residues. Indeed, integration of the RGD motif into the fiber protein of replication-deficient adenoviral vectors (see, eg, Koivunen et al., Biotechnology, 13, 265 (1995)) increases transduction of tumor cells with low CAR expression and It has been observed that when gene transfer into non-target organs after internal administration is reduced and the adenoviral vector encodes TNF-α, it exhibits potent antitumor activity in a peritoneal cancer model.

Alternatively or in addition, a replication-deficient or conditionally replicating adenoviral vector contains a chimeric coat protein comprising a non-native amino acid sequence that binds to αvβ6 integrin. αvβ6 integrin is almost or completely absent on normal epithelium and endothelium and is upregulated in several cancers including lung, colon, and ovarian cancer. Incorporation of the αvβ6 integrin-binding motif, RTDLXXL (SEQ ID NO: 1) (where X can be any amino acid) into the adenovirus fiber protein results in the adenoviral vector of the resulting adenoviral vector to cancer cells displaying αvβ6 integrin. Increases specificity and allows for therapeutically significant levels of gene expression in the target tumor tissue. Although not limited, the αvβ6 integrin-binding motif of the foot-and-mouth disease virus (FMV; Jackson et al., J. Virol., 74, 4949-4956 (2000)), the LAP-1 amino acid sequence (Munger et al., Cell, 96, 319 -328 (1999)), and RXDL (SEQ ID NO: 2) and RX ₁ DLX ₁ X ₁ X ₂ (SEQ ID NO: 3) (where X ₁ can be any amino acid, X ₂ is L, I, F, Other αvβ6 integrin-binding motifs comprising the amino acid sequence described in Kraft et al., J. Biol. Chem., 274, 1979-1985 (1999), including Y, V, or P) are adenoviruses. It can be used as a non-native amino acid sequence that is incorporated into the coat protein.

  Tumors often contain tumor cells, vasculature, and a heterogeneous mass of tumor matrix. The interstitial tumor matrix consists of collagen, glycosaminoglycans (GAGs), and proteoglycans. In order to target replication-deficient or conditional replication adenoviral vectors to tumor cells, the adenoviral coat protein of the replication-deficient or conditional replication adenoviral vector contains a non-native amino acid sequence that preferentially binds to the tumor matrix. obtain. Suitable non-native amino acid sequences include, for example, collagen bindings such as WREPSFAMLS (SEQ ID NO: 4) and WREPGRMELN (SEQ ID NO: 5) described in Hall et al., Human Gene Therapy, 11, 983-993 (2000). And other tumor matrix binding motifs identified by display technology (eg, retroviral display libraries). Replication-deficient or conditionally-replicating adenoviral vectors targeted to tumor matrix components gather in the vicinity of tumor cells, thereby increasing the likelihood of tumor cell transduction.

  In other embodiments, the adenoviral vector comprises a chimeric viral coat protein that is not selective for a particular type of eukaryotic cell. The chimeric shell protein has a non-native amino acid sequence attached by inserting a non-native amino acid sequence into or instead of the inner shell protein sequence, or at the N- or C-terminus of the shell protein By doing so, it differs from the wild-type outer shell protein. For example, a ligand comprising about 5 to about 9 lysine residues (preferably 7 lysine residues) is attached to the C-terminus of the adenovirus fiber protein via a non-coding spacer sequence. In this embodiment, the chimeric virus coat protein binds efficiently to a wider range of eukaryotic cells than the wild type virus coat, as described in International Patent Application WO 97/20051. Such adenoviral vectors may be preferred in some embodiments, since tumors do not contain a homogeneous population of cancer cells.

  Of course, the ability of adenoviral vectors to recognize potential host cells can be regulated without genetic manipulation of the coat protein, i.e. through the use of bispecific molecules. For example, complexing an adenovirus with a bispecific molecule containing a penton-based binding domain and a domain that selectively binds to a specific cell surface binding site allows the targeting of adenoviral vectors to specific cell types. It becomes possible.

  Suitable adenoviral vector mutations are U.S. Pat. 2001/0047081 A1, 2002/0099024 A1, and 2002/0151027 A1, and international patent applications WO 96/07734, WO 96/26281, WO 97/20051, WO 98/07865, WO 98/07877, WO 98/40509, WO 98/54346, WO 00/15823, WO 01/58940, and WO 01/92549.

  To further enhance the persistence of replication-deficient or conditionally-replicating adenoviral vectors in the bloodstream, the adenoviral fiber protein is made less capable of interacting with the innate or acquired host immune system Can be modified. For example, one or more amino acids of a native fiber protein can be mutated to resemble a recombinant fiber protein that is less capable of being recognized by neutralizing antibodies than wild type fiber (see, eg, International Patent Application WO 98/40509). (See Crystal et al.)). The fiber can also be modified to lack one or more amino acids that mediate interaction with the reticuloendothelial system (RES). For example, the fiber can be modified to lack one or more glycosylation or phosphorylation sites, the fiber (or virus containing the fiber) can be produced in the presence of an inhibitor of glycosylation or phosphorylation, or the adenovirus surface can be Can be mutated to lack a putative heparin sulfate proteoglycan binding domain (eg, Dechecchi et al., Virology, 268, 382-390 (2000) and Dechecchi et al., J. Virol., 75, 8772-8780 ( 2001)).

  Alternatively, or in addition, a replication-deficient or conditionally-replicating adenoviral vector immunizes on its surface to “mask” adenoviral particles from antibody recognition and other mammalian defense / clearance mechanisms such as RES. (See, for example, Moghimi and Hunter, Critical Reviews in Therapeutic Drug Carrier Systems, 18 (6), 537-550 (2001)). Inactive molecules ideally avoid the immune system, neutralizing antibodies, and other blood-derived proteins, scavenger cells, and the reticuloendothelial system. Inactive molecules also promote resistance to degrading enzymes. Immunologically inert molecules include but are not limited to poloxamer, poloxamine, poly (acrylamide), poly (2-ethyl-oxazoline), poly [N- (2-hydroxylpropyl) methylacrylamide], Examples include poly (vinyl alcohol), poly (vinyl pyrrolidone), poly (lactide-co-glycolide), poly (methyl methacrylate), poly (butyl-2-cyanoacrylate), or poly (ethylene glycol) (PEG). For PEG, virion proteins have primary amine groups, epoxy groups, or diacylclycerol groups to reduce their removal by collectin and / or opsonin affinity or RES Kupffer cells or other cells. Can be conjugated to lipid derivatives of PEG containing (eg, Kilbanonov et al., FEBS Lett., 268, 235 (1990), Senior et al., Biochem. Biophys. Acta., 1062, 11 (1991), Allen et al Biochem. Biophys. Acta., 1066, 29 (1991) and Mori et al., FEBS Lett., 284, 263 (1991)). The binding of immunologically inactive molecules to the viral surface is known in the art. For example, PEGylation of adenovirus is described in Croyle et al., J. Virol., 75 (10), 4792-4801 (2001), and US Pat. No. 6,399,385 (Croyle et al.). Several variants of PEG molecules that utilize different amino acids (eg lysine or cysteine) for attachment to the viral surface are commercially available. In order to facilitate and control the binding of PEG molecules to the viral surface, the adenoviral coat protein can be modified to include such attachment sites. Accordingly, it is appropriate that the replication-deficient or conditionally replicating adenoviral vector of the method of the invention contains one or more cysteine and / or lysine residues that are genetically incorporated into the coat protein. It may also be advantageous to incorporate a non-native amino acid sequence into the adenovirus shell to target a replication-deficient or conditionally-replicating adenoviral vector to target cells. Such non-native amino acid sequences preferably do not contain attachment sites for PEG molecules, which could lead to blockage of cell surface binding sites on non-native amino acid ligands. Thus, in one embodiment, the replication-deficient or conditionally-replicating adenoviral vector is PEGylated and the non-native amino acid sequence can cause the PEG molecule to attach to the non-native amino acid sequence and prevent cell transduction. Contains no cysteine or lysine. This construction strategy allows PEGylation of virus particles while retaining activity.

Foreign nucleic acid A replication-deficient or conditionally replicating adenoviral vector comprises at least one foreign nucleic acid. Any nucleic acid that is not native to an adenoviral vector is “foreign”. An exogenous nucleic acid encodes a peptide that exerts a biological effect in a host cell, such as, for example, a peptide associated with or treating a biological disorder. The exogenous nucleic acid can be obtained from any source, for example, isolated from natural sources, synthetically produced, or isolated from a genetically engineered organism.

  In one embodiment of the invention, the replication defective or conditionally replicating adenoviral vector comprises a nucleic acid sequence encoding TNF-α. While other members of the TNF family of proteins, such as Fas ligand and CD40 ligand, are useful in the treatment of many diseases, TNF-α has been proven to be an effective anticancer agent. The effect of TNF-α on cancer is multiple, including induction of apoptosis and tumor necrosis. TNF-α induces adhesion of vascular endothelium to neutrophils and platelets and decreases thrombomodulin production (Koga et al., Am. J. Physiol., 268, 1104-1113 (1995)). The result is clot formation in tumor vasculogenesis followed by hemorrhagic necrosis of the tumor. The nucleic acid sequence encoding TNF-α is described in detail in US Pat. No. 4,879,226. Adenoviral vectors encoding human TNF are further described in US Pat. No. 6,579,522.

The foreign nucleic acid can encode an angiogenic peptide. “Angiogenic peptides” are involved in any process leading to the formation of new blood vessels such as basement membrane disruption, cell proliferation, cell migration, vessel wall maturation, lumen formation, vasodilation, production of mediators, vessel branching, etc. Peptide. Suitable angiogenic peptides for use in the methods of the invention include, but are not limited to, endothelial mitogen, factors associated with endothelial migration, factors associated with vessel wall maturation, factors associated with vessel wall dilation, and extracellular matrix degradation. Or a transcription factor. Endothelial mitogens include, for example, vascular endothelial growth factor (VEGF, eg, VEGF ₁₂₁ , VEGF ₁₄₅ , VEGF ₁₆₅ , VEGF ₁₈₉ , VEGF ₂₀₆ , VEGF-II, and VEGF-C), fibroblast growth factor (FGF, eg, , AFGF, bFGF, and FGF-4), platelet-derived growth factor (PDGF), placental growth factor (PLGF), angiogenin, hepatocyte growth factor (HGF), tumor growth factor-beta (TGF-β), connective tissue These include growth factor (CTGF) and epidermal growth factor (EGF). Endothelial migration can be induced, for example, by Del-1. Factors associated with vessel wall maturation include, but are not limited to, angiopoietin (Ang, eg, Ang-1 and Ang-2), tumor necrosis factor-alpha (TNF-α), midkine (MK), COUP- TFII, and heparin binding neurotrophic factor (HBNF, also known as heparin binding growth factor). Vascular wall dilators include, for example, nitric oxide synthase (eg, eNOS and iNOS) and monocyte chemotactic protein-1 (MCP-1). Extracellular matrix degradation is facilitated by, for example, Ang-2, TNF-α, and MK. Suitable transcription factors include, for example, HIF-1a and PR39. Other pro-angiogenic factors include activin binding protein (ABP) and tissue inhibitors of metalloproteinases (TIMP). Coagulation factors such as tissue factor, FVIIa, FXa, thrombin, and activators of PAR1, PAR2, and PAR3 receptors are also thought to play a role in angiogenesis (eg, Carmeliet et al., Science, 293 1602 (2001)). Additional pro-angiogenic factors are described in published US Patent Application No. US2003 / 0027751 A1.

  Angiogenesis-promoting factors are described in US Pat. Nos. 5,194,596 (Tischer et al.), 5,219,739 (Tischer et al.), 5,240,848 (Keck et al.), 5,332,671 (Ferrara et al.), 5,338,840 (Bayne et al.), 5,532,343 ( Bayne et al.), 5,169,764 (Shooter et al.), 5,650,490 (Davis et al.), 5,643,755 (Davis et al.), 5,879,672 (Davis et al.), 5,851,797 (Valenzuela et al.), 5,843,775 (Valenzuela et al.), and 5,821,124 (Valenzuela et al.); International patent applications WO 95/24473 (Hu et al.) and WO 98/44953 (Schaper); European Patent Document 0 476 983 (Bayne et al.), 0 506 477 (Bayne et al.), And 0 550 296 (Sudo et al.); Japanese Patents 1038100, 2117698, 2279698, and 3178996; J. Folkman et al., Nature, 329, 671 (1987); Fernandez et al , Circulation Research, 87, 207-213 (2000), and Moldovan et al., Circulation Research, 87, 378-384 (2000). Preferably, at least one nucleic acid sequence encodes a tissue specific angiogenic factor, most preferably an endothelium specific angiogenic factor such as VEGF.

  Alternatively, the foreign nucleic acid can encode an angiogenesis inhibitor that inhibits or reduces neovascularization in a mammal. Angiogenesis inhibitors can inhibit, for example, cell proliferation, cell migration, angiogenesis, extracellular matrix degradation, mediator production, and the like. Angiogenesis inhibitors can also be antagonists to pro-angiogenic agents, thus neutralizing pro-angiogenic factors (eg, Sato, Proc. Natl. Acad. Sci. USA, 95, 5843-5844 ( 1998)).

  Angiogenesis inhibitors suitable for use in the methods of the invention include, for example, anti-angiogenic factors, cytotoxins, apoptotic factors, antisense molecules specific for angiogenic factors, ribozymes, receptors for angiogenic factors (eg, Soluble VEGF-R1 (flt-1), soluble VEGF-R2 (flk / kdr), soluble VEGF-R3 (flt-4), and VEGF receptor chimeric protein (Aiello, Proc. Natl. Acad. Sci., 92, 10457 (1995))), antibodies that bind to angiogenic factors, and antibodies that bind to receptors for angiogenic factors. Examples of the anti-angiogenic factors include angiostatin, thrombospondin, protamine, vasculostatin, endostatin, platelet factor 4, heparinase, interferon (eg, INFα) and the like. One skilled in the art will appreciate that any anti-angiogenic factor can be modified or truncated and retain anti-angiogenic activity. As such, active fragments of anti-angiogenic factors (ie, those fragments that have sufficient biological activity to inhibit angiogenesis) are suitable for use in the methods of the invention. Anti-angiogenic factors are described in US Patent 5,840,686; International Patent Applications WO 93/24529 and WO 99/04806; Chader, Cell Different., 20, 209-216 (1987); Dawson et al., Science, 285, 245-248. (1999); and Browser et al, J. Biol. Chem., 275, 1521-1524 (2000).

  Many cytotoxins and apoptotic factors are known in the art, such as p53, Fas, Fas ligand, Fas-related protein with a death domain (FADD), caspase-3, caspase-8 (FLICE), FAIM, Gax, SARP-2, caspase-10, Apo2L, IkB, DIkB, ICH-1 / CED-3-homologous protein (RAIDD) related to receptor interacting protein (RIP) , TNF-related apoptosis-inducing ligand (TRAIL), DR4, DR5, Bcl-2 cell death-inducing coding sequence including N-terminal deletion, Bcl-x coding sequence including N-terminal deletion inducing cell death, Bax , Bak, Bid, Bad, Bik, Bif-2, c-myc, Ras, Raf, PCK kinase, AKT kinase, Akt / PI (3) -kinase, PITSLRE, death-related protein (DAP) kinase, RIP, JNK / SAPK, Daxx, NIK, MEKK1, ASK1, PKR, and their variants (eg, their Dominant negative mutants and dominant positive mutants thereof), and fragments thereof (eg, their active domains), and combinations thereof. Apoptotic, cytotoxic and cytostatic transcription factors include, for example, the E2F transcription factor and its synthesis cell cycle independent, AP1 transcription factor, AP2 transcription factor, SP transcription factor (eg, SP1 transcription factor) , Helix-loop-helix transcription factors, DP transcription factors (eg, DP1, DP2, and DP3), and variants thereof (eg, their dominant negative variants and their dominant positive variants), and Fragments thereof (eg, their active domains), and combinations thereof. Apoptotic, cytotoxic and cytostatic viral proteins include, for example, adenovirus E1A product, adenovirus E4 / ORF6 / 7 product, adenovirus E4 / ORF4 product, cytomegalovirus (CMV) product (eg, CMV-thymidine kinase (CMV-TK)), herpes simplex virus (HSV) products (eg HSV-TK), human papillomavirus (HPV) products (eg HPVX), and variants thereof (eg their dominant negative) Variants and their dominant positive variants), and fragments thereof (eg, their active domains), and combinations thereof. Cytotoxins and apoptotic factors are particularly useful for inhibiting cell proliferation, an important angiogenic process. Suitable cytotoxins and apoptotic factors can be identified using conventional techniques such as, for example, cell proliferation assays and TUNEL assays, respectively.

  The foreign nucleic acid can also encode a pigment epithelium-derived factor (PEDF) or a therapeutic fragment thereof. PEDF, also referred to as early population doubling factor-1 (EPC-1), is a secreted protein that has homology with a family of serine protease inhibitors called serpins. PEDF is made mostly by retinal pigment epithelial cells and is detectable in most tissues and cell types of the body. PEDF has both neurotrophic and anti-angiogenic properties and is therefore useful in the treatment and research of a wide variety of diseases. Neurotrophic factors are thought to be responsible for the maturation of growing neurons and the maintenance of mature neurons. It is hypothesized that neurotrophic factors can actually reverse neuronal degradation associated with, for example, blindness. Neurotrophic factors function both paracrine and autocrine, making them ideal therapeutic agents. In this regard, PEDF has been observed to induce differentiation in retinoblastoma cells and enhance the survival of neural populations (Chader, Cell Different., 20, 209-216 (1987)). PEDF further has gliastatic activity, ie the ability to inhibit glial cell proliferation. PEDF also has anti-angiogenic activity. Anti-angiogenic derivatives of PEDF include the SLED protein discussed in international patent application WO 99/04806. PEDF has also been postulated to be involved in cellular senescence (Pignolo et al., J. Biol. Chem., 268 (12), 8949-8957 (1998)). PEDF is further characterized in US Pat. Nos. 5,840,686, 6,319,687, and 6,451,763, and international patent applications WO 93/24529, 95/33480, and WO 99/04806. Viral vectors containing foreign nucleic acids encoding PEDF are further described in International Patent Application WO 01/58494.

  Alternatively or additionally, the exogenous nucleic acid may encode a cytokine or chemokine. Cytokines are biological factors that regulate cell-cell interactions, cell signaling, and other cellular activities that are generally released by cells. Cytokines include, for example, interferons, interleukins, and lymphokines. Chemokines induce and promote cell migration. Cytokines include, for example, macrophage colony stimulating factor (eg, GM-CSF), interferon alpha (IFN-α), interferon beta (IFN-β), interferon gamma (IFN-γ), interleukin (IL-1, IL -2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, IL-16, and IL-18), proteins of the TNF family, Intercellular adhesion molecule-1 (ICAM-1), lymphocyte function-related antigen-3 (LFA-3), B7-1, B7-2, FMS-related tyrosine kinase 3 ligand, (Flt3L), vasoactive intestinal peptide ( VIP), and CD40 ligand. Chemokines include, for example, B cell-induced chemokine-1 (BCA-1), fractalkine, melanoma growth stimulating activity protein (MGSA), hemofiltrate CC chemokine 1 (HCC-1), interleukin 8 (IL8 ), Interferon-stimulated T cell alpha migration factor (I-TAC), lymphotactin, monocyte migration protein 1 (MCP-1), monocyte migration protein 3 (MCP-3), monocyte migration protein 4 (MCP-4), Examples thereof include macrophage-derived chemokine (MDC), macrophage inflammatory protein (MIP), platelet factor 4 (PF4), RANTES, BRAK, eotaxin, and Exodus 1-3. Cytokines and chemokines are generally described in the art, including the Invivogen catalog (2002), San Diego, CA.

  The exogenous nucleic acid can be a native nucleic acid or cDNA encoding the desired peptide, although modifications and mutations of the encoding nucleic acid sequence are possible and appropriate in the present invention. For example, the degeneracy of the genetic code allows substitution of nucleotides throughout the polypeptide coding region without change in the encoding polypeptide, as in the translation stop signal. Such an alternative sequence can be deduced from, for example, a known amino acid sequence of TNF-α or a nucleic acid sequence encoding TNF-α and constructed by conventional synthetic or site-directed mutagenesis techniques. Is possible. The synthetic DNA method is substantially equivalent to the method of Itakura et al., Science, 198, 1056-1063 (1977), and Crea et al., Proc. Natl. Acad. Sci. USA, 75, 5765-5769 (1978). Can be executed according to. Site-directed mutagenesis techniques are described in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (2d ed. 1989). Alternatively, the nucleic acid sequence may be a protein N or C as long as the peptide retains biological activity such as the tumoricidal activity of TNF-α as described in US Pat. It can encode a peptide with either end extended.

  Furthermore, a homologue of any peptide described herein, ie greater than about 70% identity (preferably greater than about 80% identity, more preferably greater than about 90% identity) to the protein at the amino acid level. And most preferably greater than about 95% identity) and a nucleic acid sequence encoding any peptide that exhibits the same level of activity as the desired peptide can be incorporated into a replication-deficient or conditionally-replicating adenoviral vector. . The degree of amino acid identity can be determined using any method known in the art, such as the BLAST sequence database. Furthermore, a protein homolog may be any peptide, polypeptide, or portion thereof that hybridizes to the protein at least moderately, preferably highly, under stringent conditions and retains biological activity. Typical moderately stringent conditions include: 20% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × Denhardt's solution, 10% Incubation at 37 ° C. in a solution containing dextran sulfate and 20 mg / ml denatured sheared salmon sperm DNA, overnight incubation followed by filter wash at about 37-50 ° C. in 1 × SSC, or substantially similar Conditions such as moderately stringent conditions detailed in Sambrook et al., Supra. Highly stringent conditions include, for example: (1) low ionic strength and high temperature for washing, eg, 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate (SDS) at 50 ° C. ( 2) Denaturing agents during hybridization, eg formamide, eg 50% (v / v) formamide / 0.1% ficoll / 0.1% polyvinylpyrrolidone (PVP) / containing 0.1% bovine serum albumin (BSA) Use 50 mM sodium phosphate buffer (pH 6.5) containing 750 mM sodium chloride, 75 mM sodium citrate at 42 ° C., or (3) 50% formamide, 5 × SSC (0.75 M NaCl, 0.075 M Acid sodium), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 × Denhard Using t's solution, sonicated salmon sperm DNA (50 mg / ml), 0.1% SDS, and 10% dextran sulfate, (i) 42 ° C. in 0.2 × SSC, (ii) in 50% formamide Washing conditions at 55 ° C. and (iii) 0.1 × SSC (preferably in combination with EDTA) at 55 ° C. Further details and explanations on the hybridization reaction's stringency are given, for example, in Ausubel et al., Supra.

  A nucleic acid sequence can encode a functional portion of a desired peptide, ie, any portion of a protein that retains a measurable level of the biological activity of the native full-length protein. For example, a functional TNF-α fragment produced by expression of a nucleic acid sequence of a replication-deficient or conditionally-replicating adenoviral vector can be expressed in human cells transiently transfected with a nucleic acid sequence encoding a fragment of the protein. Standard molecular biology and cell culture techniques such as assaying the biological activity of the fragments can be used to identify. The exogenous nucleic acid can also encode a fusion protein that includes, in part, a protein of interest paired with another preferably functional peptide moiety. For example, to increase the effectiveness of TNF-α to exert its biological effect in tumor cells, the foreign nucleic acid can be a ligand for a cellular receptor found in tumor cells, such as αvβ3, αvβ5, αvβ6. Or a fusion protein comprising TNF-α or a biologically active fragment thereof fused to a ligand that binds to CD13.

  The foreign nucleic acid sequence is desirably an expression cassette, ie, a specific nucleotide having the function of promoting subcloning and restoration of the nucleic acid sequence (eg, one or more restriction sites) or expression of the nucleic acid sequence (eg, polyadenylation or splice sites) Present as part of the array. The foreign nucleic acid is preferably located in the E1 region (eg, replacing all or part of the E1 region) or E4 region of the adenovirus genome. For example, the E1 region can be replaced with a promoter variable expression cassette containing a foreign nucleic acid. The expression cassette is preferably inserted in the 3'-5 'direction, for example, so that the direction of transcription of the expression cassette is reversed from that of the surrounding adjacent adenoviral genome. However, it is also suitable that the expression cassette is inserted in the 5'-3 'direction with respect to the direction of transcription of the surrounding genome. In addition to expression cassettes containing foreign nucleic acids, replication-deficient or conditionally replicating adenoviral vectors may contain other expression cassettes containing other foreign nucleic acids, which can replace any of the deleted regions of the adenoviral genome. It is. Insertion of the expression cassette into the adenovirus genome (eg, into the E1 region of the genome) can be facilitated by known methods, eg, by introducing a unique restriction site at a predetermined location in the adenovirus genome. As mentioned above, preferably all or part of the E3 region of the adenoviral vector is also deleted.

  Preferably, the foreign nucleic acid comprises a transcription termination region (in the transcription direction of the coding sequence), such as a polyadenylation sequence located 3 'to the angiogenic peptide coding sequence. Synthetic optimization sequences and polyadenylation of BGH (bovine growth hormone), polyomavirus, TK (thymidine kinase), EBV (Epstein Barr virus), and papillomavirus (including human papillomavirus and BPV (bovine papillomavirus)) Any suitable polyadenylation sequence can be used, including sequences. A preferred polyadenylation sequence is the SV40 (human sarcoma virus 40) polyadenylation sequence.

  Preferably, the exogenous nucleic acid is operably linked (ie, under its transcriptional control) to one or more promoter and / or enhancer elements, eg, as part of a promoter variable expression cassette. Techniques for operably linking sequences are well known in the art. Any suitable promoter or enhancer sequence can be used in the present invention. Suitable viral promoters include, for example, the cytomegalovirus (CMV) promoter (CMV immediate early promoter (eg, described in US Pat. Nos. 5,168,062 and 5,385,839), promoters derived from human immunodeficiency virus (HIV), such as the HIV long terminal repeat promoter) Rous sarcoma virus (RSV) promoter (RSV long terminal repeat, etc.), mouse mammary tumor virus (MMTV) promoter, HSV promoter (Lap2 promoter or herpes thymidine kinase promoter, etc. (Wagner et al., Proc. Natl. Acad. Sci., 78, 144-145 (1981)), SV40 or Epstein Barr virus promoter, adeno-associated virus promoter (p5 promoter etc.), etc. Preferably, the promoter is CMV immediate early pro Is Ta.

  Many of the promoters described above are constitutive promoters. Instead of being a constitutive promoter, the promoter may be an inducible promoter, ie a promoter that is up and / or down regulated in response to an appropriate signal. For example, expression control sequences that are up-regulated by chemotherapeutic agents are particularly useful for cancer applications (eg, chemically inducible promoters). Furthermore, expression control sequences can be upregulated by radiant energy sources or by substances that suppress cells. For example, expression control sequences can be upregulated by ultrasound, photoactivatable compounds, radio frequency, chemotherapy, cryogenic freezing. Preferred replication-deficient or conditionally replicating adenoviral vectors of the present invention comprise a chemically or radiation-inducible promoter operably linked to a foreign nucleic acid encoding TNF-α. The use of a radiation-inducible promoter allows local control of TNF-α production, for example by administration of radiation to cells or hosts containing adenoviral vectors, thereby minimizing systemic toxicity. Any suitable radiation inducible promoter can be used in the present invention. A preferred radiation-inducible promoter for use in the present invention is the early growth region-1 (EGR-1) promoter, particularly the CArG domain of the EGR-1 promoter. The region of the EGR-1 promoter that is likely to be responsible for radiation inducibility is located between nucleotides -550 bp to -50 bp. The EGR-1 promoter is described in detail in US Pat. No. 5,206,152 and International Patent Application WO 94/06916. Another suitable radiation-inducible promoter is the c-Jun promoter, which is activated by X-rays. The region of the c-Jun promoter that is likely to be responsible for radiation inducibility is believed to be located between nucleotides -1.1 kb and 740 bp. The c-Jun promoter and EGR-1 promoter are further described, for example, in US Pat. No. 5,770,581.

  The promoter can also be a tissue or cell specific promoter such as a tumor cell selective promoter. Tumor cell selective promoters suitable for replication deficient or conditionally replicating adenoviral vectors include, but are not limited to, the E2F promoter and the DF3 (muc-1) promoter. The promoter may also be selective for tumor associated endothelial cells, such as the flt-1 promoter.

Dosage and Method of Administration A dose of replication deficient or conditionally replicating adenoviral vector is gradually released into the mammalian bloodstream. The dose of replication deficient or conditionally replicating adenoviral vectors will depend on many factors, including the size of the target tissue, the degree of any side effects, the particular route of administration, and the like. Desirably, a single dose of replication defective or conditionally replicating adenoviral vector comprises at least about 1 × 10 ⁵ particles (also referred to as particle units) to at least about 1 × 10 ¹³ particles of adenoviral vector. The dose is preferably at least about 1 × 10 ⁶ particles (eg, about 4 × 10 ⁶ -4 × 10 ¹² particles), more preferably at least about 1 × 10 ⁷ particles, more preferably at least about 1 × 10 ⁸ particles ( For example, about 4 × 10 ⁸ -4 × 10 ¹¹ particles), and most preferably at least about 1 × 10 ⁹ particles to at least about 1 × 10 ¹⁰ particles (eg, about 4 × 10 ⁹ -4 × 10 ¹⁰ particles). Adenovirus vector. Alternatively, the dosage is about 1 × 10 ¹⁴ particles or less, preferably about 1 × 10 ¹³ particles or less, even more preferably about 1 × 10 ¹² particles or less, even more preferably about 1 × 10 ¹¹ particles or less, and most preferably Includes about 1 × 10 ¹⁰ particles or less (eg, about 1 × 10 ⁹ particles or less). In other words, a single dose of replication-deficient or conditionally replicating adenoviral vector is about 1 × 10 ⁶ particle units (pu), 2 × 10 ⁶ pu, 4 × 10 ⁶ pu, 1 × 10 ⁷ pu, 2 × 10 ⁷ pu, 4 × 10 ⁷ pu, 1 × 10 ⁸ pu, 2 × 10 ⁸ pu, 4 × 10 ⁸ pu, 1 × 10 ⁹ pu, 2 × 10 ⁹ pu, 4 × 10 ⁹ pu, 1 × 10 ¹⁰ pu, 2 × 10 ¹⁰ pu, 4 × 10 ¹⁰ pu, 1 × 10 ¹¹ pu, 2 × 10 ¹¹ pu, 4 × 10 ¹¹ pu, 1 × 10 ¹² pu, 2 × 10 ¹² pu, or 4 × 10 ¹² pu Or replication-defective adenoviral vectors.

The volume of the carrier that dilutes the replication-deficient or conditionally-replicating adenoviral vector, particularly the pharmaceutically acceptable carrier, is administered in the size of the mammal and usually the dose of the replication-deficient or conditional replication adenoviral vector in the pharmaceutical composition It will depend on the period. For example, if the volume of the carrier is based on the size or mass of the mammal, the dose of replication-deficient or conditionally-replicating adenoviral vector comprises about 20 ml or more physiologically acceptable carrier per kilogram (kg) of mammal. It is administered in a pharmaceutical composition. Preferably, the pharmaceutical composition comprises about 40 ml or more of a physiologically acceptable carrier / kg of mammal, more preferably about 60 ml or more of a physiologically acceptable carrier / kg of mammal. . Even more preferably, the pharmaceutical composition comprises about 80 ml or more of a physiologically acceptable carrier / kg of mammal, and most preferably about 100 ml or more of a physiologically acceptable carrier / mammalian. Includes kg. Alternatively, the volume of a pharmaceutical composition administered to a mammal can be calculated based on the surface area of the mammal, a method routinely used in pharmacology. In this regard, the pharmaceutical composition comprises about 75 ml or more (eg, about 100 ml or more) of a physiologically acceptable carrier per square meter of mammalian surface area. Preferably, the pharmaceutical composition is about 150 ml or more (eg, about 175 ml or more, about 200 ml or more, or about 250 ml or more) of a physiologically acceptable carrier / mammalian surface area m ^2. including. More preferably, the dose of replication defective or conditionally replicating adenoviral vector is in a pharmaceutical composition comprising 275 ml or more (eg 300 ml or more) of a physiologically acceptable carrier / mammalian surface area m ^2. Is administered. It will be appreciated that smaller volume carriers may be suitable in some embodiments, for example, as described in US Patent Application Publication 2003/0086903.

  A dose of replication deficient or conditionally replicating adenoviral vector is gradually released into the mammalian bloodstream. “Slowly released” means that a single dose of replication-deficient or conditionally replicating adenoviral vector is released into the mammalian bloodstream over at least about 15 minutes. Sustained release of replication-deficient or conditionally replicating adenovirus doses allows a larger fraction of the adenoviral vector dose to circulate in the mammalian bloodstream than previously achieved, thereby reducing replication deficiency or Increases the likelihood that a conditionally replicating adenoviral vector will reach the target tissue. In one embodiment, the dose of replication defective or conditionally replicating adenovirus is continuously released into the bloodstream for at least about 30 minutes (eg, at least about 45, 60, 90, 120, or 150 minutes). Preferably, the dose of replication-deficient or conditionally replicating adenoviral vector is at least about 3 hours (eg, at least about 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7 .5, 8, 8.5, 9, or 9.5 hours). Also preferably, the dose of replication deficient or conditionally replicating adenoviral vector is administered to the mammal over at least about 10 hours.

  Sustained release into the mammalian bloodstream can be achieved by various routes of administration as known to those skilled in the art. A dose of replication deficient or conditionally replicating adenoviral vector can be released directly into the systemic circulation by intravenous or intraarterial administration. Other devices can be used to facilitate sustained release, as the use of a syringe may not be desirable for administration of replication deficient or conditionally replicating adenovirus doses over at least about 15 minutes. For example, intravenous infusion and delivery catheter devices with storage tanks, infusion pumps, etc. are particularly suitable for slow release of substances into the systemic circulation. Similarly, many sustained release implants are suitable for delivering replication deficient or conditionally replicating adenoviral vectors into the bloodstream. Microparticles for sustained release of substances in the body are often composed of biodegradable polymers that release a calculated amount of the therapeutic as the microparticles degrade. Sustained release formulations can include, for example, gelatin, chondroitin sulfate, polyphosphate esters (eg, bis-2-hydroxyethyl terephthalate (BHET)), or poly (lactic acid-glycolic acid). Sustained release devices and formulations are further described, for example, in US Patents 5,378,475, 5,629,008, 5,733,567, 6,506,410, and 6,455,526.

  Instead of releasing the dose of replication-deficient or conditionally replicating adenoviral vector directly into the bloodstream, replication deficiency is achieved by introducing the replication-deficient or conditionally replicating adenoviral vector into a mammalian region that gradually flows out into the circulatory system. Alternatively, a dose of the conditional replicating adenoviral vector can be administered indirectly into the blood stream, so that the dose of replication deficient or conditional replicating adenovirus is released into the blood stream over at least about 15 minutes. One means of such indirect systemic delivery involves administering a dose of an adenoviral vector to the lymphatic system. The function of the lymph is partly maintaining the body's fluid balance. The lymphatic system collects fluid from tissues and returns interstitial fluid to the bloodstream in the thoracic duct. Administration of doses of replication deficient or conditionally replicating adenoviral vectors to the lymphatic system takes advantage of the body's natural and steady release of substances into the bloodstream.

  Many methods for introducing doses of replication-deficient or conditionally replicating adenoviral vectors into the lymph, such as those known to those skilled in the art, are suitable for use in the methods of the invention. For example, the abdominal cavity is the main source of drainage to the lymphatic system. Parenteral or intraperitoneal delivery of doses of replication deficient or conditionally replicating adenoviral vectors is one method of administration into the bloodstream via the lymph. The dose of replication deficient or conditionally replicating adenoviral vector can be delivered to the peritoneal cavity using any suitable means such as infusion or instillation.

  Before administering a dose of a replication-deficient or conditionally replicating adenoviral vector containing a foreign nucleic acid, administering a “pre-dose” of a substance that saturates the mammalian natural innate clearance mechanism, such as an adenoviral vector Can be advantageous. The pre-dose can include any adenovirus or adenovirus vector construct described herein, and preferably has a reduced ability to transduce mesothelial cells or hepatocytes than wild-type adenovirus vectors of the same serotype. A replication-deficient or conditionally-replicating adenoviral vector. While not wishing to be bound by any particular theory, administration of a pre-dose of an adenoviral vector prevents single-dose replication deficient or conditionally replicating adenoviruses by interfering with or interacting with mammalian clearance effector cells. It is believed to increase the persistence of the vector, thereby allowing a larger fraction of a single dose of replication deficient or conditionally replicating adenoviral vectors to reach the bloodstream and remain in circulation. Alternatively or additionally, a pre-dose of adenoviral vector can cause tolerance to replication deficient or conditionally replicating adenoviral vectors in mammals. The pre-dose of the adenoviral vector is any suitable in any suitable volume of physiologically acceptable carrier, such as the adenoviral vector dose described herein and the volume of the physiologically acceptable carrier. May contain any number of adenoviral particles. Similarly, a pre-dose of an adenoviral vector can be administered to a mammal using any route of administration, such as intravenous, intra-arterial, or intra-peritoneal delivery, and a replication-deficient or conditionally replicating adenoviral vector dose. Can occur at any time prior to the administration of, preferably the administration of the pre-dose increases the circulation time of the dose of replication-deficient or conditionally replicating adenoviral vectors. The pre-dose is preferably administered from about 5 minutes to about 60 minutes (eg, from about 10 minutes to about 45 minutes) prior to administration of the replication deficient or conditionally replicating adenoviral vector dose. For example, the pre-dose can be administered from about 15 minutes to about 30 minutes prior to administration of a dose of a replication-deficient or conditionally replicating adenoviral vector.

Normalized mean blood flow concentration The present invention provides a method for enhancing the persistence of adenoviral vectors in the systemic circulation, thereby increasing the likelihood that a replication deficient or conditionally replicating adenovirus will contact the target tissue. The relative exposure of the target to the treatment containing the gene transfer vector can be determined by calculating the mean blood flow concentration of the treatment over a period of time. The average blood flow concentration is calculated by standard methods as shown below.

  The amount of replication-deficient or conditionally replicating adenoviral vector in the mammalian bloodstream measured at various time points (denoted “T”) after administration of the replication-deficient or conditionally replicating adenoviral vector at t = 0 ( Concentration) (expressed as “Cv” in units of [adenovirus vector particle / blood unit volume]) and plot a dose curve (Cv vs. T). The area under the resulting Cv vs. T curve (AUC) (with units of [(adenoviral vector particle / unit volume) (time)]) is relative to the target, replication deficient or conditionally replicating adenoviral vector Standard pharmacological indicator of exposure. For example, following administration of replication deficient or conditionally replicating adenoviral vectors at time = 0 minutes, adenoviral vectors in the bloodstream at 10, 30, 90, 180, 360 and 1440 minutes after administration Measure the concentration. Using the concentration of replication deficient or conditionally replicating adenoviral vectors at each time point, an adenoviral vector concentration (Cv) versus time (T) curve is plotted. The AUC can then be calculated from the plotted curve according to the following equation:

  Average blood flow concentration (Cv (ave)) expressed as replication deficient or conditionally replicating adenoviral vector particles per unit volume of blood over a period of time from t = 0 to t = T (eg, 24 hours or 1440 minutes) Is calculated by dividing AUC by T (ie, Cv (ave) = AUC / T). Cv (ave) is then normalized by expressing as a percentage of the theoretical blood flow concentration of the replication-deficient or conditionally-replicating adenoviral vector obtained as the adenoviral vector is never removed from circulation (Cv (0)) be able to. Cv (0) is obtained by dividing the vector dose (D; expressed in adenoviral vector particles) by the mammalian blood volume (Vb) (ie Cv (0) = D / Vb). Normalized mean blood flow concentration of replication deficient or conditional replication adenoviral vectors expressed as a percentage of the theoretical blood flow concentration (Cv (0)) of adenoviral vector doses that are never removed from the blood stream (Cv (ave )%) Is then calculated by dividing Cv (ave) by Cv (0) and multiplying by 100% (ie Cv (ave)% = [Cv (ave) / Cv (0)] 100%) . Cv (ave)% is a convenient indicator that compares the relative blood flow persistence of two different adenoviral vectors administered to a mammal in the same manner.

  Normalization of replication deficient or conditionally replicating adenoviral vectors over about 24 hours after administration in the blood stream expressed as a percentage of the theoretical blood flow concentration of the adenoviral vector dose that is never removed from the blood stream in the method of the present invention. The average blood flow concentration is at least about 1% (eg, at least about 2%). Preferably, the normalized mean blood flow concentration of the replication defective or conditionally replicating adenoviral vector over about 24 hours after administration in the bloodstream is at least about 3% (eg, at least about 4%), more preferably at least about 5% (Eg, at least about 6% or at least about 7%). Even more preferably, the normalized average blood flow concentration of the replication-deficient or conditionally replicating adenoviral vector over about 24 hours after administration in the bloodstream is at least about 8% (eg, at least about 9%), and most preferably at least About 10% (eg, about 11% or more).

  Alternatively, the normalized mean blood flow concentration of a replication-deficient or conditionally-replicating adenoviral vector is equal to the normalized average blood flow concentration of the wild-type adenovirus, replication-defective It is compared to an equivalent dose of an adenoviral vector of the same serotype as a conditionally replicating adenoviral vector, or an equivalent dose of an adenoviral vector having the ability of wild type adenovirus to infect mesothelial cells or hepatocytes. For example, the normalized mean blood flow concentration of the replication deficient or conditionally replicating adenoviral vector over about 24 hours after administration is preferably at least about 5 times the normalized mean blood flow concentration of the equivalent dose of the wild type adenoviral vector. Large (eg, at least about 6 times, 7 times, 8 times, or 9 times larger). More preferably, the normalized mean blood flow concentration of the replication deficient or conditionally replicating adenoviral vector over about 24 hours after administration is preferably at least about normalized blood flow concentration of the equivalent dose of the wild type adenoviral vector. 10 times larger (eg, at least about 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, or 45 times larger). Even more preferably, the normalized mean blood flow concentration of the replication deficient or conditionally replicating adenoviral vector over about 24 hours after administration is preferably at least greater than the normalized mean blood flow concentration of the equivalent dose of the wild type adenoviral vector. About 50 times larger (eg, at least about 60 times, 70 times, 80 times, 90 times, or 100 times larger).

Cancer treatment
The present invention further provides a method of destroying tumor cells in a mammal. The method involves gradual delivery of a replication-deficient or conditionally replicating adenoviral vector dose into the mammalian bloodstream. Replication-deficient or conditionally-replicating adenoviral vectors are described herein as follows: (a) a nucleic acid sequence encoding an oncogenic factor and (b) adenoviral entry via a coxsackievirus and adenoviral receptor (CAR). Including adenovirus fiber proteins that do not mediate Tumor cells and / or cells associated with or in close proximity to the tumor are transduced to produce tumoricidal factors, thereby destroying mammalian tumor cells. A number of tumoricidal factors have been described herein and have been identified in the art. A preferred tumoricidal factor is TNF-α. Ideally, the target tissue is a solid tumor or a tumor associated with soft tissue in humans (ie, soft tissue sarcoma). Tumors include oral cavity and pharynx, digestive system, respiratory system, bone and joints (eg, bone metastases), soft tissue, skin (eg, melanoma), breast, genital system, urinary system, eyes and orbits, brain and nervous system (Eg, glioma), or endocrine system (eg, thyroid or adrenal gland) (ie, located there) can be associated with a cancer and not necessarily the primary tumor. Tissues associated with the oral cavity include, but are not limited to, tongue and mouth tissue. Cancer can occur in tissues of the digestive system including, for example, the esophagus, stomach, small intestine, colon, rectum, anus, liver (eg, hepatobiliary cancer), gallbladder, pancreas. Respiratory cancers can affect the larynx, lungs, and bronchi and include, for example, non-small cell lung cancer. Tumors can occur in the cervix, uterine body, ovaries, vulva, vagina, prostate, testis, penis that make up the male and female genital system, and in the bladder, kidney, renal pelvis, ureter that make up the urinary system obtain. The target tissue can also be lymphoma (eg, Hodgkin's disease or non-Hodgkin's disease lymphoma), multiple myeloma, or leukemia (eg, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, etc.) Can be related to The recombinant gene transfer vectors and methods described herein are used in one embodiment for the treatment of ovarian cancer, so that one or more ovarian tumors are reduced in size or destroyed.

Tumors can be at any stage and can receive other treatments. The replication deficient or conditionally replicating adenoviral vectors of the methods of the invention treat tumors that have proven to be resistant to other forms of cancer treatment, such as radiation resistant tumors (ie, tumor cell destruction or tumor size). It is useful to reduce). Tumors can also be any size. The replication-deficient or conditionally-replicating adenoviral vectors of the methods of the invention mediate the reduction in tumor size (eg 42 cm ² (cross-sectional area) or 4400 cm ^{3 in} volume) that was initially large. Ideally, the method of the present invention results in cancer (tumor) cell death and / or reduction in tumor size. It will be appreciated that tumor cell death can occur without substantial reduction in tumor size due to, for example, the presence of support cells, angiogenesis, fibrous matrix, and the like. Thus, a reduction in tumor size is preferred but not essential for the treatment of cancer.

  One advantage of the method of the present invention over previous cancer treatments is that tumor cells can be targeted while better avoiding non-target tissues. Reducing native binding of replication-deficient or conditionally replicating adenoviral vectors reduces transduction of non-target tissues such as liver, spleen, kidney, and lung, thereby transducing target tissues, eg, tumors It provides a larger fraction of the dose of replication-deficient or conditionally replicating adenoviral vectors that can be used. To further enhance the efficiency of delivery of tumoricidal factors to tumor cells, replication-deficient or conditionally-replicating adenoviral vectors are like adenoviral fiber proteins specific for cell receptors expressed on tumor cells. May include non-native amino acid sequences (ie, ligands) incorporated into adenovirus coat proteins. Examples of suitable non-native amino acid sequences include, but are not limited to, non-native amino acid sequences that bind to αvβ3, αvβ5, and αvβ6 integrins. By practicing the method of the present invention, the ratio of tumor transduction levels with replication-deficient or conditionally replicating adenoviral vectors, for example, compared to the level of liver transduction with replication-deficient or conditionally replicating adenoviral vectors, It can reach at least about 0.1: 1. Preferably, the ratio of the level of tumor transduction by the replication defective or conditionally replicating adenoviral vector is at least about 0.5: 1 compared to the level of liver transduction by the replication defective or conditionally replicating adenoviral vector, Most preferably it is at least about 1: 1.

Pharmaceutical Compositions Replication deficient or conditionally replicating adenoviral vectors are desirably present in pharmaceutical compositions comprising a pharmaceutically acceptable carrier (eg, a physiologically acceptable carrier). Any suitable pharmaceutically acceptable carrier can be used in the present invention and such carriers are well known in the art. The choice of carrier will be determined in part by the particular site at which the pharmaceutical composition is administered and the particular method used to administer the pharmaceutical composition.

  Suitable formulations include isotonic sterile solutions including aqueous and non-aqueous solutions, antioxidants, buffers, bacteriostats, and solutes that are isotonic with the blood or other body fluids of the intended recipient of the formulation, And aqueous and non-aqueous sterile suspensions containing suspending, solubilizing, thickening, stabilizing, and preserving agents. Preferably, the pharmaceutically acceptable carrier is a liquid containing a buffer and a salt. The formulations can be stored in unit-dose or multiple-dose sealed containers, such as ampoules and vials, in a freeze-dried state where only a sterile liquid carrier, such as water, can be added just prior to use. Extemporaneous solutions and suspensions can be prepared from sterile powders, granules, and tablets. Preferably, the pharmaceutically acceptable carrier is a buffered saline solution.

  More preferably, the pharmaceutical composition is formulated to protect the adenoviral vector from damage prior to administration. Certain formulations desirably reduce the photosensitivity and / or temperature sensitivity of adenoviral vectors. In fact, since the pharmaceutical composition will be maintained for various periods of time, it should be formulated to ensure stability and maximum activity upon administration. Usually, the pharmaceutical composition is maintained at a temperature above 0 ° C., preferably above 4 ° C. (eg 4-10 ° C.). In some embodiments, the pharmaceutical composition may be at a temperature of 10 ° C or higher (eg, 10-20 ° C), 20 ° C or higher (eg, 20-25 ° C), or even 30 ° C or higher (eg, 30-40 ° C). It may be desirable to maintain. The pharmaceutical composition may be maintained at the above temperature for at least 1 day (eg, 7 days (1 week) or longer), but usually the period is, for example, at least 3, 4, 5 prior to administration to the patient. Or longer, such as 6 weeks, or longer, eg, at least 10, 11, or 12 weeks. During that period, the adenoviral gene transfer vector optimally loses little or no activity, but some loss of activity can be tolerated, particularly at relatively high storage temperatures and / or This is the case when the storage time is long. Preferably, the activity of the adenoviral vector composition is reduced by no more than about 20%, preferably no more than about 10%, more preferably no more than about 5% after any of the periods described above.

  For this purpose, the pharmaceutical composition is preferably a pharmaceutically acceptable liquid carrier, such as those described above, and polysorbate 80, L-arginine, polyvinylpyrrolidone, α-D-glucopyranosyl α-D-glucopyranoside diwater. Contains a stabilizer selected from the group consisting of Japanese (usually known as trehalose), and combinations thereof. More preferably, the stabilizer is trehalose or trehalose in combination with polysorbate 80. The stabilizer can be present in any suitable concentration in the pharmaceutical composition. When the stabilizing agent is trehalose, trehalose is desirably present at a concentration of about 2-10% (w / v), preferably about 4-6% (w / v) of the pharmaceutical composition. When trehalose and polysorbate 80 are present in the pharmaceutical composition, trehalose is preferably present at a concentration of about 4-6% (weight / volume), more preferably about 5% (weight / volume), but polysorbate 80 is desirably present at a concentration of about 0.001 to 0.01% (weight / volume), more preferably about 0.0025% (weight / volume). When a stabilizer, such as trehalose, is included in the pharmaceutical composition, the pharmaceutically acceptable liquid carrier preferably comprises a sugar other than trehalose. Appropriate formulation of pharmaceutical compositions is further described in US Pat. Nos. 6,225,289 and 6,514,943 and International Patent Application WO 00/34444.

  In addition, the pharmaceutical composition can include additional therapeutic or bioactive agents. For example, there may be therapeutic agents that are useful for the treatment of specific indications. Factors that control inflammation, such as ibuprofen and steroids, can become part of the pharmaceutical composition and reduce swelling and inflammation associated with in vivo administration of adenoviral vectors and physiological distress. An immune system suppressant can be administered with the pharmaceutical composition to reduce the immune response to the adenoviral vector itself or the immune response associated with the disease. Alternatively, an immunopotentiator can be included in the pharmaceutical composition to up-regulate the body's original defense against disease.

Radiation therapy The typical course of treatment for most types of cancer is radiation therapy. Accordingly, the methods of the present invention may further comprise administering a line dose of radiation to the subject. Radiation therapy eradicates cancer cells by inducing mutations in cellular DNA using high-energy particles such as X-rays or gamma rays or a beam of waves. Tumor tissue is more sensitive to radiation than normal tissue in that cancer cells divide more rapidly than normal cells. Radiation has also been shown to increase exogenous DNA expression in exposed cells. When the nucleic acid sequence encoding TNF-α is operably linked to a radiation-inducible promoter, in addition to the additive or synergistic effect of radiation and TNF-α observed when removing tumor cells, radiation therapy Continuously during the period of time, at the tumor site, radiation enhances TNF-α production and maintains therapeutic levels of TNF-α (eg, Hersh et al., Gene Therapy, 2, 124-131 (1995), and Kawashita et al. al., Human Gene Therapy, 10, 1509-1519 (1999)).

  Any type of radiation can be administered to a mammal as long as the mammal can tolerate radiation dose without significant negative side effects. Suitable types of radiation therapy include, for example, ionizing (electromagnetic) radiation therapy (eg, X-rays or gamma rays) or particle beam radiation therapy (eg, high linear energy radiation). Ionizing radiation is defined as radiation that contains particles or photons with sufficient energy to ionize (ie, to acquire or lose electrons) (eg, as described in US Pat. No. 5,770,581). The effect of radiation can be controlled at least in part by the clinician. Preferably, the radiation dose is divided for maximum target cell exposure and toxicity reduction. Radiation can be administered at the same time as a radiation-sensitive agent that enhances killing of tumor cells or a radiation protective agent that protects healthy tissue from the harmful effects of radiation (eg, IL-1 or IL-6). Similarly, the application of heat, i.e., hyperthermia, or chemotherapy, makes the tissue sensitive to radiation.

  The radiation source may be external or internal to the mammal. External radiation therapy is most common and involves, for example, using a linear accelerator to direct a beam of high energy radiation through the skin and to the tumor site. Although the beam of radiation is localized at the tumor site, it is almost impossible to avoid exposure of normal and healthy tissue. However, external radiation is usually well tolerated by the patient. Internal radiation therapy involves the implantation of radiation sources such as beads, wires, pellets, capsules, etc. into the body at or near the tumor site. Such implants can be removed after treatment or can remain inert in the body. Types of internal radiation therapy include, but are not limited to, brachytherapy, interstitial radiation therapy, intracavitary radiation therapy. A less common form of internal radiation therapy is radioimmunotherapy, in which a tumor-specific antibody conjugated to a radioactive substance is administered to a patient. The antibody locates and binds to the tumor antigen, thereby efficiently administering a single dose of radiation to the relevant tissue.

  Whatever the mode of administration, preferably the total dose of radiation administered to the mammal in the present invention is from about 5 Gray (Gy) to about 70 Gy. More preferably, about 10 Gy to about 65 Gy (eg, about 15 Gy, 20 Gy, 25 Gy, 30 Gy, 35 Gy, 40 Gy, 45 Gy, 50 Gy, 55 Gy, or 60 Gy) is administered over a period of treatment. Although the total dose of radiation can be administered over a period of one day, ideally the total dose is divided and administered over several days. Desirably, radiation therapy is at least about 3 days, such as at least 5, 7, 10, 14, 17, 21, 25, 28, 32, 35, 38, 42, 46, 52, or 56 days (about 1- 8 weeks). Thus, the daily dose of radiation is approximately 1-5 Gy (eg, about 1 Gy, 1.5 Gy, 1.8 Gy, 2 Gy, 2.5 Gy, 2.8 Gy, 3 Gy, 3.2 Gy, 3.5 Gy, 3.8 Gy). 4Gy, 4.2Gy, or 4.5Gy), preferably 1-2Gy (eg, 1.5-2Gy). If operably linked to a radiation-inducible promoter, the daily dose of radiation should be sufficient to induce expression of the nucleic acid sequence. If the period is extended, radiation is preferably not administered daily, thereby resting the subject and realizing a therapeutic effect. For example, desirably, radiation is administered on 5 consecutive days for each week of treatment and not on 2 days, thereby allowing for 2 days of rest per week. However, depending on the patient's response to treatment and possible side effects, radiation may be 1 day / week, 2 days / week, 3 days / week, 4 days / week, 5 days / week, 6 days / week, or Can be administered on all 7 days / week.

Chemotherapy Like radiation, chemotherapy is a standard treatment for reducing tumor size or destroying tumors. A dose of one or more chemotherapeutic agents can be administered to the mammal in conjunction with administration of a replication-deficient adenoviral vector comprising a nucleic acid sequence encoding TNF-α. The chemotherapeutic agent can be administered prior to administering the replication-deficient adenoviral vector, after administering the replication-deficient adenoviral vector, or in the same pharmaceutical composition or as a separate administration at the same time as the replication-deficient adenoviral vector. Any suitable chemotherapeutic agent can be used. Suitable chemotherapeutic drugs include, but are not limited to, adriamycin, asparaginase, bleomycin, busulfan, cisplatin, carboplatin, carmustine, capecitabine, chlorambucil, cytarabine, cyclophosphamide, camptothecin, dacarbazine, dactinomycin, daunorubicin, dexrazoxane, docetaxel , Doxorubicin, etoposide, floxuridine, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine, mechlorethamine, mercaptopurine, melphalan, methotrexate, mitomycin, mitoxantrone, mitoxantrone nitrosurea), paclitaxel, pamidronate, pentostatin, Pricamycin, procarbazine, rituximab, streptozocin, teniposide, thioguanine, thiotepa, vinblastine, vincristine, vinorelbine, taxol, transplatinum, anti-vascular endothelial growth factor compound ("anti-VEGF"), anti-epidermal growth factor receptor compound ("Anti-EGFR"), 5-fluorouracil and the like. The type and number of chemotherapeutic agents administered to a subject will depend on the standard chemotherapeutic regimen for the particular tumor type. In other words, certain cancers may generally be treated with a single chemotherapeutic agent, while others may be commonly treated with a combination of chemotherapeutic agents. Preferably, the chemotherapeutic agent administered to the subject is selected from the group consisting of 5-fluorouracil (5-FU), cisplatin, paclitaxel, gemcitabine, cyclophosphamide, capecitabine, and / or doxorubicin. Any suitable dose of one or more chemotherapeutic agents can be administered to a mammal, eg, a human. Suitable chemotherapeutic drug doses as described above are well understood in the art and are described, for example, in US Patent Application Publication No. 2003/0082685 A1. In embodiments where a dose of 5-FU is administered to a human patient, the dose is preferably from about per ^{m 2} of the patient's body surface area per day 50 mg (i.e., mg / ^{m 2} / day) to about 1500 mg / ^{m 2} / Days (eg, about 100 mg / m ² / day, about 500 mg / m ² / day, and about 1000 mg / m ² / day). More preferably, the dose of 5-FU is about 100 mg / m ² / day to about 300 mg / m ² / day (eg, 200 mg / m ² / day) or about 900 mg / m ² / day to about 1100 mg / m ^2. / Day (eg, about 1000 mg / m ² / day). When a dose of cisplatin is administered to a human patient, the dose is preferably about 25 mg / m ² / day to about 500 mg / m ² / day (eg, about 50 mg / m ² / day, about 100 mg / m ² / day, Or about 300 mg / m ² / day). More preferably, the dose of cisplatin about 50-100 mg / ^{m 2} / day, most preferably 75 mg / ^{m 2} / day. When a dose of capecitabine is administered to a patient, the dose is preferably about 500 mg / m ² / day to about 1500 mg / m ² / day (eg, about 700 mg / m ² / day, about 800 mg / m ² / day, or About 900 mg / m ² / day). More preferably, the dose of capecitabine comprises about 800 mg / m ² / day to about 1000 mg / m ² / day (eg, about 900 mg / m ² / day).

  Similar to radiation, if the period is extended, chemotherapy is not administered daily, thereby resting the subject and realizing a therapeutic effect. For example, desirably, chemotherapy is administered on 5 consecutive days for each week of treatment and not on 2 days, thereby allowing for 2 days of rest per week. However, depending on the patient's response to treatment and possible side effects, chemotherapy may be 1 day / week, 2 days / week, 3 days / week, 4 days / week, 5 days / week, 6 days / week, Or it can be administered every 7 days / week.

In some embodiments, it may be advantageous to use a method of administration of one or more chemotherapeutic agents in which the dose is administered continuously to the subject over an extended period of time. For example, continuous infusion of chemotherapeutic drugs into a subject may be desirable. In this regard, the duration of administration of the dose of one or more chemotherapeutic agents can be any suitable length of time. Standard infusion rates for the chemotherapeutic agents described herein are known in the art and can be modified in any suitable manner depending on the nature of the disease. For example, when 5-FU is administered, the standard infusion rate is about 96 hours per treatment week (ie, 5 days per week). Other aspects and dosing schedules for cancer chemotherapy, for example, Bast et al. (Eds. ), Cancer Medicine, 5 th edition, BC. Decker Inc., Hamilton, are described in Ontario (2000).

(Example)
The following examples further illustrate the invention but, of course, should not be construed as limiting its scope in any way.

Example 1
This example demonstrates that adenoviral vectors administered to mammals according to the method of the present invention persist for a prolonged period in the circulation.

An adenovirus serotype 5 vector was created in which most of the coding sequences of the E1 and E3 regions of the adenovirus genome were deleted. The replication-deficient adenoviral vector includes a luciferase reporter gene operably linked to a cytomegalovirus (CMV) promoter (AdL). In order to reduce adenoviral fiber-mediated transduction through CAR, the AB loop of the adenoviral fiber protein was modified to disrupt CAR binding (AdL.F ^* ). To further reduce cell surface interactions with native adenovirus, the integrin binding domain of adenovirus penton base protein was disrupted (AdL.F ^* PB ^* ). Methods for constructing and propagating AdL, AdL.F ^* , and AdL.F ^* PB ^* and adenoviral vectors with reduced natural tropism are described in Einfeld et al., J. Virol., 75, 11284-11291 (2001). Is further described.

C57B1 / 6 mice anesthetized by inhalation of 2-4% isoflurane were administered intravenously via the jugular vein at a dose of 1 × 10 ¹¹ particles of AdL, AdL.F ^* , or AdL.F ^* PB ^* . Effective viral load in the bloodstream was quantified at 10, 60, 180, and 1440 minutes after administration. At each time point, the percentage of injected dose was measured and graphed as a function of time after administration of the vector (see FIG. 1). The area under the resulting curve (AUC) and the normalized mean blood flow concentration of each adenoviral vector was calculated as described herein. The resulting data is shown in Table 1, and the normalized mean blood flow concentration of AdL and AdL.F ^* PB ^* at each time point is expressed as “% AUC”.

  At 24 hours after administration (ie, 1440 minutes), the normalized mean blood flow concentration (“% AUC”) was less than 1% for both adenoviral vector constructs.

Other mouse populations received a dose of 1 × 10 ¹¹ particles of AdL, AdL.F ^* , or AdL.F ^* PB ^* in 500 μl composition intraperitoneally. The amount of virus present in the bloodstream was quantified 90, 180, 360, and 1440 minutes after administration. At each time point, the percentage of infused dose (“% dose”) was measured and graphed as a function of time after administration of the vector (see FIG. 2). The normalized mean blood flow concentrations for AdL, AdL.F ^* , and AdL.F ^* PB ^* are calculated as described herein and shown in Table 2, and the normalized mean blood concentration is “% AUC”. Represent as

Approximately 24 hours after administration (ie, 1440 minutes), approximately 0.0004% of the injected dose of AdL was present in the circulation. The normalized average blood flow concentration (“% AUC”) of AdL at 24 hours is approximately 0.022%, ie significantly less than 1%. In 24 hours, normalized average bloodstream concentration of AdL.F ^* is approximately 2.1%, AdL.F ^* PB ^* normalized average blood flow concentration was approximately 0.05%. The normalized mean blood flow concentration of AdL.F ^* at 24 hours was approximately 97 times that of AdL compared to AdL whose adenovirus shell was not modified. The normalized mean blood flow concentration of AdL.F ^* PB ^* was approximately 2.2 times that of AdL.

  This example shows that intraperitoneal administration of adenoviral vectors modified to reduce innate binding to host cell receptors as a route of delivery to the systemic circulation reduces the clearance of such vectors from the bloodstream. Make it clear.

(Example 2)
This example demonstrates that preadministration of an adenoviral vector to a mammal can increase the persistence of the dose of replication-deficient adenoviral vector in the circulation.

Three populations of mice were anesthetized with 2-4% isoflurane via inhalation, pre-dose AdNull of 2 × 10 ¹¹ particles, a fiber and penton protein lacking its reporter gene and disrupting its native cell surface binding site E1 / E3-deficient adenovirus containing was administered. After 10 minutes (t = 0), a dose of 1 × 10 ¹¹ particles in 500 μl of physiologically acceptable carrier was administered, one of the three adenoviral vector constructs described in Example 1. . The amount of adenovirus vector in the circulation was recorded. At each time point, the percentage of injected dose was measured and graphed as a function of time after administration of the vector (see FIG. 3). The normalized mean blood flow concentrations for AdL, AdL.F ^* , and AdL.F ^* PB ^* are calculated as described herein and shown in Table 3, with the normalized mean blood concentration as “% AUC” To express.

In comparison with the data shown in Table 2, administration of a pre-dose of adenoviral vector increased the half-life of the adenoviral vector in the bloodstream with all three adenoviral vector constructs. The largest increase in circulation time was observed with the double-removed adenoviral vector AdL.F ^* PB ^* and enjoyed a 210-fold increase in normalized mean blood flow concentration.

In another study, C57Bl / 6 mice under 2-4% isoflurane anesthesia were treated with vehicle (10 mM Tris / HCl (pH 7.8) buffer containing 5% trehalose, 10 mM MgCl ₂ , and 150 mM NaCl), 1 ×. Purified adenovirus hexon protein equivalent to the amount of hexon protein present in a 100 μl composition of 10 ¹¹ adenovirus particles, or a pre-dose AdNull of 2 × 10 ¹¹ particles in 100 μl composition intraperitoneally Administered. Ten minutes later (t = 0), a dose of 1 × 10 ¹⁰ or 1 × 10 ¹¹ particles of AdL.F ^* PB ^* in 100 μl of composition was administered intraperitoneally as described in Example 1. The amount of AdL.F ^* PB ^* in the bloodstream was measured at various times after vector administration. At each time point, the ratio to the injected dose was measured and graphed as a function of time after administration of the vector (see FIG. 4). The normalized mean blood flow concentration of AdL.F ^* PB ^* is calculated as described herein and shown in Table 4, and the normalized mean blood flow concentration is expressed as “% AUC”.

  Pre-administration of hexon protein had no detectable effect on vector persistence in the bloodstream beyond that seen with vehicle pre-administration. Pre-administration of AdNull increased the normalized mean blood flow concentration of the dose of both replication-deficient adenoviral vectors administered. At 24 hours after administration, pre-administration increased the normalized mean blood flow concentration by at least approximately 800-fold compared to the blood flow concentration of the same adenoviral vector administered without a pre-dose of adenoviral vector. This result also suggests that increasing the dose and volume of the composition results in maximal persistence of the adenoviral vector in the circulation.

  The data given in this example supports that administration of a pre-dose of adenoviral vector can further increase the circulation time of the therapeutic adenoviral vector dose in the bloodstream.

(Example 3)
This example illustrates how to modify an adenoviral vector to further increase the half-life in circulation.

The virus surface of AdL.F ^* PB ^* described in Example 1 was coated with PEG molecules. Specifically, AdL.F ^* PB ^* was desalted by passing the adenoviral vector through a DG column equilibrated with 10 mM potassium phosphate buffer containing 10% sucrose. AdL.F ^* PB ^* (9 × 10 ¹² particles, 0.25 mg protein) in the ratio of 1: 5 and 1:50 by adding 1 mg / ml mPEG-succinimidyl propionate (MW = 5000) solution (Adenovirus protein weight: PEG reagent weight). The PEGylation reaction was stopped by adding an excess amount of 10X lysine. The PEGylated virus buffer was replaced with 10 mM Tris / HCl (pH 7.8) containing 5% trehalose, 150 mM NaCl, and 10 mM MgCl ₂ by passing the vector through a DG column.

AdL, AdL.F ^* PB ^* , AdL.F ^* PB ^* (PEG-5), or AdL.F ^* PB ^* (PEG-50) dose (1 diluted in 500 μl physiologically acceptable carrier X10 ¹¹ pu adenoviral vector) was injected intraperitoneally into mice anesthetized with 2-4% isoflurane. The amount of adenoviral vector in the bloodstream was measured at various times after administration. At each time point, the ratio to the injected dose was measured and graphed as a function of time after administration of the vector. Normalized mean blood flow concentrations for AdL, AdL.F ^* PB ^* , AdL.F ^* PB ^* (PEG-5), and AdL.F ^* PB ^* (PEG-50) are calculated as described herein. The normalized average blood flow concentration is expressed as “% AUC”.

  PEGylation of doubly-removed adenoviral vectors increased adenoviral vector retention in the bloodstream by at least a factor of two. The higher the concentration of PEG molecules attached to the virus surface, the further increased the half-life of the adenoviral vector. These results demonstrate that adenoviral particle surface masking reduces adenoviral vector dose clearance when administered according to the methods of the present invention.

Example 4
This example illustrates that the method of the invention can efficiently deliver an adenoviral vector containing a transgene to tumor tissue in vivo.

Of NCI-H441 tumor-bearing nude mice, a clinically relevant subcutaneous tumor-bearing animal model, out of four E1 / E3-deficient adenoviral vector constructs containing a luciferase reporter gene all operably linked to a CMV promoter One of the doses was administered. AdL and AdL.F ^* PB ^* are described in Example 1. A ligand that binds to αvβ3 and αvβ5 integrin and mediates viral transduction was inserted into the adenovirus fiber protein HI loop of AdL.F ^* PB ^* to produce AdL ^** RGD. A ligand that binds to αvβ6 (SEQ ID NO: 1) was inserted into the HI loop of the adenovirus fiber protein of AdL.F ^* PB ^* to prepare AdL ^** αvβ6. Mice were anesthetized via inhalation of 2-4% isoflurane prior to adenoviral vector administration.

Two administration methods were used to deliver doses of adenoviral vectors. A portion of the mice was administered intravenously with a dose of 1 × 10 ¹¹ particles of adenovirus vector diluted in 100 μl of a pharmaceutically acceptable carrier. The remaining mice were given a pre-dose AdNull of 2 × 10 ¹¹ particles as described in Example 2 intraperitoneally 10 minutes prior to receiving a replication-deficient adenoviral vector at a dose of 1 × 10 ¹¹ particles via intraperitoneal injection. Injected into. Tumor, liver, spleen, kidney, and / or lung tissue were collected 24 hours after administration of AdL, AdL.F ^* PB ^* , AdL ^** RGD, or AdL ^** αvβ6. The total protein amount in the sample was measured by the Bio-Rad protein assay, and the amount of luciferase activity was measured by luminescence and expressed as relative light units (RLU) per milligram of total protein. The intensity of luciferase expression was used to quantify adenoviral vector transduction (see FIGS. 5 and 6). The ratio of tumor transduction to other tissue transduction was calculated and summarized in Table 6.

  The ratio of tumor transduction compared to that of other tissues was normalized by comparing the level of AdL transduction. Table 7 shows the normalized data.

  This example demonstrates that the method of the present invention substantially increases the delivery of gene transfer vectors to tumor tissue over intravenous delivery and provides an alternative to direct injection of gene transfer vectors into tumors To do. Modifying adenoviral vectors to reduce native binding to cell surface receptors increases the level of tumor tissue transduction compared to liver transduction and non-native to adenoviral fiber protein Insertion of the ligand further enhances targeting to tumor tissue while avoiding other non-target tissues.

  All references, including publications, patent applications, and patents cited herein, are individually and specifically indicated that each reference is incorporated herein by reference, and To the same extent as described in its entirety, it is incorporated herein by reference.

  In the context of describing the invention (especially in the context of the appended claims), the use of the terms “a” and “an” and “the” and like directives is specifically referred to herein. Unless explicitly stated or otherwise clearly contradicted by context, this should be interpreted to encompass both singular and plural. The terms “comprising”, “having”, “including” and “containing” are open-ended terms (ie, including “to” unless otherwise specified). Means “but not limited”). The description of a range of values herein is merely intended to serve as an abbreviation method for individually referring to each separate value within the range, unless otherwise specified herein. , And each separate value is intended to be included herein as if it were individually listed herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples or exemplary words (e.g., "such as") provided herein are merely intended to better clarify the invention and are specifically claimed otherwise. Unless otherwise specified, the scope of the present invention is not limited. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

  Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors anticipate that those skilled in the art will use such variations as appropriate, and that the inventors have implemented the invention in a state different from that specifically described herein. Intended to be. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

FIG. 1 is a graph of percentage vs. injected dose of AdL and AdL.F ^* PB ^* after intravenous adenoviral vector injection. FIG. 2 is a graph of percentage vs. injected dose of AdL, AdL.F ^* , and AdL.F ^* PB ^* after intraperitoneal injection of adenovirus vector. FIG. 3 is a graph of percentage vs. injected dose of AdL, AdL.F ^* , and AdL.F ^* PB ^* after intraperitoneal injection of adenovirus vector. Ten minutes prior to administration of the adenoviral vector, a pre-dose of empty adenoviral vector was administered. FIG. 4 shows injection of 1 × 10 ¹⁰ particle units (pu) or 1 × 10 ¹¹ pu AdL or AdL.F ^* PB ^* with or without a pre-dose of empty adenoviral vector (Null) after vector injection Is a graph of% versus dose versus dose. FIG. 5 shows relative in samples taken from tumor, liver, spleen, kidney, and lung tissue and generated by intraperitoneal delivery of AdL, AdL.F ^* PB ^* , AdL. ^** RGD, or AdL. ^** αvβ6. FIG. 2 is a bar graph showing target light units (RLU) / mg protein. FIG. 6 is relative to samples taken from tumor, liver, spleen, kidney, and lung tissue and generated by intravenous delivery of AdL, AdL.F ^* PB ^* , AdL. ^** RGD, or AdL. ^** αvβ6. FIG. 2 is a bar graph showing target light units (RLU) / mg protein.

Claims (53)

A method for expressing a foreign nucleic acid in a mammal, wherein the method has a reduced ability to transduce mesothelial cells and hepatocytes compared to a wild-type adenovirus, and a replication-deficient or conditionally-replicating adenovirus vector containing the foreign nucleic acid A gradual release of a dose of
At least about 1 normalized mean blood flow concentration of replication deficient or conditionally replicating adenovirus over 24 hours after administration, expressed as a percentage of the initial theoretical blood flow concentration of adenoviral vector dose never removed from the bloodstream %
A method of transducing host cells in mammals to express foreign nucleic acids. 2. The method of claim 1, wherein the normalized average blood flow concentration of the replication deficient or conditionally replicating adenovirus over 24 hours after administration is at least about 3%. 3. The method of claim 1 or 2, wherein the normalized mean blood flow concentration of replication deficient or conditionally replicating adenovirus over 24 hours after administration is at least about 5%. 4. The method of any of claims 1-3, wherein the normalized mean blood flow concentration of the replication deficient or conditionally replicating adenovirus over 24 hours after administration is at least about 8%. 5. The method of any of claims 1-4, wherein the normalized average blood flow concentration of the replication deficient or conditionally replicating adenovirus over 24 hours after administration is at least about 10%. A method for expressing a foreign nucleic acid in a mammal, wherein the method has a reduced ability to transduce mesothelial cells and hepatocytes compared to a wild-type adenovirus vector, and is a replication-deficient or conditionally-replicating adenovirus containing a foreign nucleic acid Gradually delivering a dose of the vector into the mammalian bloodstream,
A method wherein the normalized mean blood flow concentration of the replication-deficient or conditionally replicating adenoviral vector over 24 hours after administration is at least about 5 times greater than the normalized mean blood flow concentration of the equivalent dose of the wild-type adenoviral vector. 7. The normalized mean blood flow concentration of a replication deficient or conditionally replicating adenoviral vector over 24 hours after administration is at least about 10 times greater than the normalized mean blood flow concentration of an equivalent dose of a wild type adenoviral vector. Method. The normalized mean blood concentration of a replication-deficient or conditionally replicating adenoviral vector in the blood stream over 24 hours after administration is at least about 50 times greater than the normalized mean blood concentration of an equivalent dose of a wild-type adenoviral vector. The method according to 6 or 7. The method according to any of claims 1 to 8, wherein the native binding of the replication-deficient or conditionally-replicating adenoviral vector to Coxsackievirus and adenoviral receptor (CAR) is reduced. 10. The method according to any of claims 1 to 9, wherein the replication-deficient or conditionally replicating adenoviral vector comprises a fiber protein in which the native CAR binding site is disrupted. 11. A method according to any of claims 1 to 10, wherein the native binding of the replication deficient or conditionally replicating adenoviral vector to integrin is reduced. 12. The method according to any of claims 1 to 11, wherein the replication-deficient or conditionally replicating adenoviral vector comprises a penton base protein in which the native integrin binding site is disrupted. 13. The method of any of claims 1-12, wherein the method comprises releasing a dose of a replication defective or conditionally replicating adenoviral vector into the bloodstream for at least about 15 minutes. 14. The method of any of claims 1-13, wherein the method comprises releasing a dose of a replication defective or conditionally replicating adenoviral vector into the bloodstream for at least about 3 hours. 15. The method of any of claims 1-14, wherein the method comprises releasing a dose of a replication defective or conditionally replicating adenoviral vector into the bloodstream for at least about 10 hours. 13. A method according to any of claims 1 to 12, wherein a dose of replication defective or conditionally replicating adenoviral vector is delivered to the bloodstream via the lymph. 13. A method according to any of claims 1 to 12, wherein the dose of replication deficient or conditionally replicating adenoviral vector is administered intraperitoneally. 18. The method of claim 17, wherein the method comprises administering a pre-dose of a replication-deficient or conditionally replicating adenoviral vector prior to administering a dose of the replication-deficient or conditionally replicating adenoviral vector. 19. The method of claim 18, wherein the pre-dose of replication deficient or conditionally replicating adenoviral vector is administered intravenously. 19. The method of claim 18, wherein a pre-dose of replication deficient or conditionally replicating adenoviral vector is administered intraperitoneally. 21. The method of any of claims 1-20, wherein the replication-deficient or conditionally replicating adenoviral vector comprises a chimeric coat protein comprising a non-native amino acid sequence that binds to a cellular receptor. 24. The method of claim 21, wherein the chimeric shell protein comprises at least a portion of an adenovirus fiber protein. 23. The method of claim 21 or 22, wherein the chimeric shell protein further comprises a spacer. 24. A method according to any of claims 21 to 23, wherein the non-native amino acid sequence is incorporated into the exposed loop of the adenovirus fiber protein. 24. A method according to any of claims 21 to 23, wherein the non-native amino acid sequence is located at the C-terminus of the adenovirus fiber protein. Replication-deficient or conditionally-replicating adenoviral vectors have poloxamer, poloxamine, poly (acrylamide), poly (2-ethyl-oxazoline), poly [N- (2-hydroxypropyl) methylacrylamide], poly (vinyl alcohol) on the surface , Poly (vinyl pyrrolidone), poly (lactide-co-glycolide), poly (methyl methacrylate), poly (butyl-2-cyanoacrylate) or poly (ethylene glycol) (PEG). The method in any one of. 27. The method of claim 26, wherein the one or more cysteine and / or lysine residues are genetically incorporated into the coat protein of a replication-deficient or conditionally-replicating adenoviral vector. 27. A method according to any of claims 21 to 26, wherein the replication-deficient or conditionally replicating adenoviral vector is PEGylated and the non-native amino acid sequence does not contain lysine. 27. A method according to any of claims 21 to 26, wherein the replication-deficient or conditionally replicating adenoviral vector is PEGylated and the non-native amino acid sequence does not contain cysteine. 30. The method according to any of claims 1 to 29, wherein the replication-deficient or conditionally-replicating adenoviral vector is deficient in a gene function essential for replication of one or more of the E1 and E4 regions of the adenoviral genome. The method according to any one of claims 1 to 30, wherein the host cell is a tumor cell. A replication-deficient or conditionally-replicating adenoviral vector comprises a chimeric adenoviral fiber protein comprising a non-native amino acid sequence attached to the C-terminus of an adenoviral fiber protein via a spacer, wherein the non-native amino acid sequence is present on tumor cells 32. The method of any one of claims 1-31, wherein the method binds to a tumor cell receptor. 35. The method of claim 32, wherein the non-native amino acid sequence binds to αvβ6 integrin on tumor cells. 35. The method of claim 32, wherein the non-native amino acid sequence binds to αvβ3 and / or αvβ5 integrin expressed in tumor cells. 35. The method of claim 32, wherein the tumor is associated with a tumor matrix and a non-native amino acid sequence binds to the tumor matrix. A medicament comprising a replication-deficient or conditionally replicating adenoviral vector dose of 20 ml or more physiologically acceptable carrier / mammalian kg or 75 ml or more physiologically acceptable carrier / mammalian surface area m ² 36. The method according to any of claims 1-35, wherein the method is administered in a composition. A medicament comprising a replication-deficient or conditionally replicating adenoviral vector dose of 100 ml or more of a physiologically acceptable carrier / kg of mammal or 300 ml or more of a physiologically acceptable carrier / mammalian surface area m ² 37. The method according to any of claims 1-36, wherein the method is administered in a composition. A method for destroying tumor cells in a mammal comprising: (a) a nucleic acid sequence encoding an oncolytic factor; and (b) an adenovirus fiber protein that does not mediate adenovirus entry via coxsackievirus and adenovirus receptors (CAR). A method comprising gradually delivering a replication-deficient or conditionally-replicating adenoviral vector into the bloodstream, thus producing tumorigenic factors and destroying tumor cells in a mammal. 40. The method of claim 38, wherein the replication deficient or conditionally replicating adenoviral vector has a reduced ability to transduce mesothelial cells and hepatocytes compared to wild type adenovirus. 40. The method of claim 38 or 39, wherein a dose of replication deficient or conditionally replicating adenoviral vector is delivered to the bloodstream via the lymph. 40. The method of claim 38 or 39, wherein a dose of replication deficient or conditionally replicating adenoviral vector is delivered to the bloodstream via intraperitoneal administration. 42. The method of any of claims 38-41, wherein the native binding of the replication-deficient or conditionally replicating adenoviral vector to the integrin is reduced. 43. The method of any of claims 38-42, wherein the normalized mean blood flow concentration of the replication deficient or conditionally replicating adenoviral vector over 24 hours after administration is at least about 1%. 44. The method of any of claims 38-43, wherein the normalized mean blood flow concentration of the replication deficient or conditionally replicating adenoviral vector over 24 hours after administration is at least about 3%. 45. The method of any of claims 38-44, wherein the normalized average blood flow concentration of the replication defective or conditionally replicating adenoviral vector over 24 hours after administration is at least about 8%. 47. The method of any of claims 38-46, wherein the replication-deficient or conditionally replicating adenoviral vector comprises a chimeric coat protein comprising a non-native amino acid sequence that binds to a cell surface receptor expressed in a tumor. 48. The method of claim 46, wherein the non-native amino acid sequence binds to αvβ6 integrin on tumor cells. 48. The method of claim 46, wherein the non-native amino acid sequence binds to αvβ3 and / or αvβ5 integrin. 49. The method of claim 46, wherein the tumor is associated with the tumor matrix and the non-native amino acid sequence binds to the tumor matrix. 39. The ratio of tumor transduction levels with replication-deficient or conditionally replicating adenoviral vectors is at least about 0.1: 1 compared to the level of liver transduction with replication-deficient or conditionally replicating adenoviral vectors. 50. The method according to any one of -49. The ratio of the level of tumor transduction by a replication-deficient or conditionally replicating adenoviral vector is at least about 0.5: 1 compared to the level of liver transduction by a replication-deficient or conditionally replicating adenoviral vector. 50. The method according to any one of 50. 52. The ratio of tumor transduction levels due to replication-deficient or conditionally replicating adenoviral vectors is at least about 1: 1 compared to liver transduction levels due to replication-deficient or conditionally replicating adenoviral vectors. A method according to any one of the above. 53. The method according to any of claims 38 to 52, wherein the tumoricidal factor is tumor necrosis factor-alpha (TNF-α).

Images